Modified Streptococcus Pneumoniae Pneumolysin (PLY) Polypeptides

ABSTRACT

This disclosure relates to immunogenic compositions comprising mutant  Streptococcus pneumonia  pneumolysin (PLY) proteins. Nucleic acids, polypeptides encoded thereby, compositions containing the same, methods for using such nucleic acids, polypeptides and compositions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.14/269,501 filed May 5, 2014, now U.S. Pat. No. 8,951,531B2, which is acontinuation application of U.S. Ser. No. 13/140,894 filed Aug. 5, 2011,now U.S. Pat. No. 8,758,766B2, which was filed under 35 U.S.C. §371 andclaims priority to International Application No. PCT/CA2009/001843,filed Dec. 22, 2009, which claims priority to U.S. provisionalapplication 61/140,804 filed Dec. 24, 2008. These applications areincorporated herein by reference in their entirety.

TECHNICAL FIELD

This disclosure relates to immunogenic compositions comprising modifiedStreptococcus pneumoniae pneumolysin (PLY) polypeptides. Nucleic acids,polypeptides encoded thereby, compositions containing the same, methodsfor using such nucleic acids, polypeptides and compositions are alsoprovided.

BACKGROUND

Streptococcus pneumoniae is an important pathogen, causing invasivediseases such as pneumonia, meningitis, and bacteraemia. Even in regionswhere effective antibiotic therapy is freely available, the mortalityrate from pneumococcal pneumonia can be as high as 19% in hospitalizedpatients. In developing countries, in excess of three million childrenunder the age of five years die each year from pneumonia, of which S.pneumoniae is the most commonly identified causative agent. S.pneumoniae also causes less serious, but highly prevalent infectionssuch as otitis media and sinusitis, which have a significant impact onhealth-care costs in developed countries. Otitis media is especiallyimportant in young children, while sinusitis affects both children andadults.

Currently, licensed anti-pneumococcal vaccines are based on formulationsof various capsular polysaccharide antigens derived from highlyprevalent strains. Serotypes that most commonly cause invasivepneumococcal infection appear to differ somewhat in various regions ofthe world. In North America in the pre-vaccination era, serotypes 4, 6B,9V, 14, 18C, 19F and 23F were the seven most common serotypes causinginvasive disease in children aged ≦5 years of age (Butler, et al. J.Infect. Dis. 171 (4): 855-889 (1995)). These serotypes were reported tobe responsible for 70-88% of invasive disease in these children andaccounted for 100% of S. pneumoniae with high-levelpenicillin-resistance.

Two types of pneumococcal vaccines are in clinical use: the 23-valentPneumococcal Polysaccharide Vaccine (23-PPV) and the 7-valentPneumococcal Conjugate Vaccine (PCV7) (Siber, et al. PneumococcalVaccines: The Impact of Conjugate Vaccine. Washington D.C.: ASM Press;2008). The polysaccharide antigens in 23-PPV elicit a T-cell-independentimmune response, resulting in poor immunologic memory. Additionally,while 23-PPV confers 60-80% protection against invasive pneumococcaldisease (IPD) in adults and the elderly, immunity wanes substantiallyafter 5 years and it is poorly immunogenic in children≦2 years of age.Robust T-cell responses with immunologic memory is observed in youngchildren vaccinated with PCV7 (Prevnar®, Wyeth Pharmaceuticals, Inc.)Prevnar 13 (PCV13), which includes the serotypes of PCV7 and serotypes1, 3, 5, 6A, 7F, and 19A was recently recommended for approval by a Foodand Drug Administration advisory committee on vaccines and relatedbiological products. Studies are also underway on a ten-valent vaccine(Synflorix, GSK) containing polysaccharides of ten pneumococcalserotypes (1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F) conjugated to acarrier protein.

Despite their resounding success and significant public health impact,anti-pneumococcal conjugate vaccines have also had some well knownshortcomings, including the complexity of conjugate vaccine production,which increases manufacturing costs. More important, however, is thefinding that polysaccharide-based conjugate vaccines protect onlyagainst infections caused by bacteria that express the particularcapsule-type represented by the vaccine. This is a problem in regionssuch as Latin America, Asia and Africa, in which the serotypesrepresented by PCV7 are responsible for much less invasive disease thanelsewhere in the world (Black, et al. In: Plotkin S A, et al. eds.Vaccines, 5th Edition. WB Saunders. Chapter 23 (2008); Garcia, et al.Rev. Panam. Salud. Publica. 19(5):340-8 (2006); Lagos, R. Pediatr.Infect. Dis. J. 21 (12): 1115-23 (2002)). This need may be addressed bythe new generation of conjugate vaccines, the 10- and 13-valent PCVvaccines (PCV-10 and PCV-13), currently in development and/or licensurestage.

Regional issues (e.g., limited serotype coverage, the potential forreplacement disease with non-vaccine serotypes, capsular switching,carrier-induced suppression, and manufacturing and supply constraintsare understood by those of skill in the art to present significantproblems in vaccinating worldwide populations. It is known that S.pneumoniae is antigenically and clonally diverse (Hanage, et al. Infect.Immun. 73(1):431-5 (2005)), with a single pneumococcal serotypetypically including a number of genetically divergent clones.Pneumococcal proteins are known to be much more conserved betweenserotypes and have thus been considered as potential vaccines.

Pneumolysin has been reported to be an intracellular protein that causesa variety of toxic effects in vivo when released upon lysis ofpneumococci. The protein is highly conserved in both amino acid sequenceand antigenicity among clinical isolates, thus satisfying some basiccriteria for its use as a vaccine antigen (Paton, et al. Infect. Immun.40(2) 548-52 (1983); Lock, et al. Microb. Pathog. 5(6): 461-67 (1988)).However, it has inherent hemolytic properties, and mutants havetherefore been developed and studied for their potential as vaccines.Historically the most commonly studied pneumolysin mutant is PdB,containing a single amino acid change, Trp433Phe (Paton, et al. Infect.Immun. 59(7):2297-304 (1991); Lu, et al. Infect. Immun. 77(5): 2076-83(2009); Ogunniyi, et al. Infect Immun. 75(1):350-7 (2007); Berry, et al.Infect. Immun. 63(5): 1969-74 (1995); Berry, et al. Infect. Immun.67(2): 981-85 (1999)).

Other PLY mutants, including ΔAla146 with a deletion of amino acid 146,and ΔAla146R147, have been recently described (Kirkham, et al. Infect.Immun. 74(1): 586-93 (2006)). Both ΔAla146 and ΔAla146R147 were shown tolack haemolytic activity against human erythrocytes. Alum-adjuvantedΔAla146 was also shown to be as protective as alum-adjuvanted wild-typePLY.

Although there were differences in the mouse strains used, the S.pneumoniae serotypes used and the routes of immunization, compilation ofresults from these studies using sepsis models have indicated that PdBor ΔAla146 prolong survival of mice when compared with placebo controlgroups. In the pneumonia model, immunization of mice with PdB yielded asignificant decrease in numbers of pneumococci in the lungs of infectedmice compared to a placebo control (Briles, et al. J. Infect. Dis.188(3): 339-48 (2003)). Moreover, in most of these findings, PdB wasfound to provide a superior protection against a wide variety of strainswhen used in combination with other virulence factors such as PspA, PspCor PsaA (Lu, supra; Ogunniyi, supra; Berry (1995), supra). Although thePdB mutant provided significant protection in some models, it had thedrawback of possessing residual hemolytic activity. However, asmentioned above, ΔAla146 provided both protective immunity and a lack ofhaemolytic activity.

While several mutant PLY vaccines have been developed, there is a clearneed in the art for additional mutants that are both protective and lackhaemolytic activity. One such mutant, PlyD1, is described herein. Thedata presented herein shows that PlyD1 lacks hemolytic activity,generates neutralizing antibodies that inhibit hemolysis by PLY invitro, and is protective in certain animal models.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Exemplary PLY amino acid sequences—alleles and modifications.

FIG. 2. In vitro hemolytic activity, toxin mediated lysis of sheep redblood cells, of mutations at position 293.

FIG. 3. Decreased lung tissue damage mediated by PLY followingimmunization with mPLY.

FIG. 4. PlyD1 protein purification based on SDS-PAGE analysis.

FIG. 5. Survival of CBA/J mice immunized 3× with different doses ofAdjuvanted Ply-D1.

SUMMARY OF THE DISCLOSURE

This disclosure relates to modified Streptococcus pneumoniae pneumolysinproteins (“mPLY”) or fragments thereof which can be used as immunogensin an immunogenic composition or vaccine against invasive pneumococcaldisease caused by S. pneumoniae. The mPLY typically include at least oneamino acid difference from wild-type PLY (“wtPLY”; e.g., any of SEQ IDNOS.:2-42). Upon administration of mPLY to a host, mPLY induces theproduction of anti-wtPLY and/or anti-mPLY antibodies, which may includeantibodies that prevent and/or inhibit the hemolytic activity of wtPLYand/or protective antibodies against organisms expressing wtPLY.Preferably, mPLY induces antibodies reactive with wtPLY and exhibitsdecreased hemolytic activity (including none) against erythrocytes ascompared to wtPLY. In some embodiments, wtPLY is modified at one or moreof amino acid 65 (threonine, “Thr65”), amino acid 293 (glycine,“Gly293”), and/or 428 (cysteine, “Cys428”) to any other amino acid. Incertain embodiments, wtPLY is modified at Gly293 and one or moreadditional amino acids such as, but not limited to, Thr65 and/or Cys428.In certain embodiments, Thr65 is substituted by cysteine, Gly293 issubstituted by cysteine, and/or Cys428 is substituted by alanine. Alsoprovided are nucleic acids encoding mPLY, methods for making mPLY,methods for immunizing, protecting, treating, and/or preventing hostsfrom infection or infected by organisms expressing wtPLY, andcompositions for doing the same. Other embodiments will be clear fromthe descriptions provided herein.

DETAILED DESCRIPTION

Provided herein are modified pneumolysin (PLY) polypeptides andfragments thereof, immunogenic compositions, and vaccines comprisingsuch polypeptides and/or fragments (e.g., peptides), methods ofpreparing such polypeptides and/or fragments, and methods of using suchpolypeptides and/or fragments. A modified PLY polypeptide (“mPLY”)and/or fragment of mPLY is one having differences in nucleic acid oramino acid sequence as compared to a wtPLY sequence. A mPLY polypeptidetypically, but not necessarily, has at least one modified amino acidsequence as compared with a wtPLY and/or fragment thereof. Themodification is typically an amino acid substitution, insertion, and/ordeletion. The mPLY polypeptide and/or fragment thereof may be preferablysubstantially nontoxic when compared to a protein substantiallycorresponding to a wtPLY protein at the same dose. Preferably, themodified sequences provide for changes in certain activities of mPLYthat are typically associated with wtPLY, especially undesirableactivities including but not limited to membrane permeation, cell lysis,and cytolytic activity against human erythrocytes and other cells (a“cell” may be human and/or non-human). It is also preferred that mPLYretain the ability to induce anti-PLY (including but not limited toanti-wtPLY) protective and/or neutralizing antibodies followingadministration to a host (e.g., an animal such as a mammal, e.g., humanbeing). In certain embodiments, such antibodies reduce (includingeliminate) the hemolytic activity of wtPLY, and/or lung tissue toxicity,and/or bind to microorganisms expressing wtPLY, and/or bind to wtPLY perse, and/or provide protection against infection or dissemination ofdisease caused by microorganisms expressing wtPLY (e.g., Streptococcuspneumoniae).

The wtPLY sequence may be any PLY expressed in an organism, includingbut not limited to a microorganism pathogenic (e.g., “causing disease”)in a higher organism (e.g, an animal such as a mammal, e.g., humanbeing). An exemplary pathogenic organism is Streptococcus pneumoniae.Wild-type PLY polypeptides are typically encoded by a ply gene, whichare highly conserved genes but do exhibit some variation (e.g., Walker,et al. Infect. Immun. 55: 1184-1187 (1987); Mitchell, et al. NucleicAcid Res. 18: 4010 (1990); Jefferies, et al. J Infect Dis. 196(6):936-44(2007)). A PLY structural model was constructed based on sequencesimilarity with perfringolysin O and homology modeling using theDiscovery Studio suite of programs available from Accelrys Software,Inc. The model indicates that the wt-PLY is ˜20% α-helix and ˜40%β-sheet with four distinct structural domains: D1 (residues 6-21,58-147, 198-243, 319-342), D2 (residues 22-57, 343-359), D3 (residues148-197, 244-318), and D4 (residues 360-469). Wild-type PLY may bemodified in any one or more of D1, D2, D3, or D4. Examplary wtPLY aminoacid sequences include but are not limited to those shown in any ofWalker, et al. (supra), Mitchell, et al. (supra), Jeffries, et al.(supra), FIG. 1, SEQ ID NOS.: 2-42, GenBank Accession Nos. M17717,EF413923, EF413924, EF413925, EF413926, EF413927, EF413928, EF413929,EF413930, EF413931, EF413932, EF413933, EF413934, EF413935, EF413936,EF413937, EF413938, EF413939, EF413940, EF413941, EF413942, EF413943,EF413944, EF413945, EF413946, EF413947, EF413948, EF413949, EF413950,EF413951, EF413952, EF413953, EF413954, EF413955, EF413956, EF413957,EF413958, EF413959, and/or EF413960. It is noted that any of thevariations between these sequences may be combined with any othervariations found within such wtPLY (which may include syntheticmodifications described herein or elsewhere) to generate additionalwtPLY that may be modified to generate mPLY. Such wtPLY polypeptidestypically, but not necessarily, share at least about (referring to eachof the following values independently) 90%, 95%, or 99% sequenceidentity with the wtPLY amino acid sequence shown in SEQ ID NO.:2 asdetermined using mafft (Kato, et al. Nucleic Acids Res. 30: 3059-3066(2002); Kato, et al. Brief Bioinform. 9: 286-294 (2008)). In certainembodiments, a wtPLY may share 98.8% sequence identity with the wtPLYsequence shown in SEQ ID NO.:2 as determined using mafft. Themodifications introduced into wtPLY to produce mPLY may be made to anypolypeptide sharing identity with wild-type PLY, including thosepolypeptides having about 90%, 95%, 98%, 99%, or 99.9% identify with SEQID NO.:2. Any differences in the amino acid sequence of wtPLYpolypeptides are typically but not necessarily phenotypically silent. Itshould be noted that the wtPLY listed herein are only exemplary; othersuitable wtPLY sequences are known in the art and would be suitable tomodification as described herein.

Exemplary mPLY polypeptides contain, for example, wtPLY modified atamino acids threonine 65 (“Thr65”), glycine 293 (“Gly293”), and/orcysteine 428 (“Cys428”). A modification of any of these amino acids maybe referred to using conventional nomenclature, e.g., by indicating theamino acid being modified and the modification made thereto (e.g.,Thr65X, Gly293X, Cys428X, where X typically indicates the substitutedamino acid, or a deletion of that amino acid). In one embodiment, wtPLYis modified at Thr65 to cysteine (“Ply-T65C”). In another embodiment,wtPLY is modified at glycine 293 to alanine (“Ply-G293A”), cysteine(“Ply-G293C”), valine (“Ply-G293V”), or threonine (“Ply-G293T”). Inanother embodiment, wtPLY is modified at Cys428 to alanine(“Ply-Cys428A”). In other embodiments, wtPLY is modified at Gly293 andone or more additional amino acids such as, but not limited to, Thr65and/or Cys428. In other embodiments, wtPLY is modified at Thr65 and oneor more additional amino acids such as, but not limited to, Gly293and/or Cys428. In other embodiments, wtPLY is modified at Cys428 and oneor more additional amino acids such as, but not limited to, Thr65 and/orGly293. Modifications at Thr65, Gly293, and/or Cys428 may be made to anywtPLY to produce mPLY polypeptides. Preferred substitutions in each ofthese embodiments is cysteine for Thr65; cysteine, valine, or threoninefor Gly293; and/or alanine for Cys428. Exemplary mPLY polypeptides areshown in SEQ ID NOS. 43 and 44. Any of these modifications may beintroduced into any wtPLY described herein or elsewhere combined withany other modifications to produce mPLY polypeptides using standardmolecular biology and biochemical techniques.

PLY (wtPLY or mPLY) activity may be assayed and characterized by methodsknown to those of skill in the art (e.g., Paton, et al., supra; Nato, etal. Infect Immun. 59(12):4641-4646 (1991)). Any one or more of theassays described herein, or any other one or more suitable assays, maybe used to determine the suitability of mPLY as a vaccine. Two suitable,exemplary assays are the in vitro hemolytic assay and the hemolysisinhibition assay. The in vitro hemolysis assay measures the hemolytic(e.g., cytolytic) activity of mPLY relative to wtPLY. The hemolysisinhibition assay measures the ability of antisera raised against mPLY toinhibit hemolysis by PLY, and (typically) comparing anti-mPLY antiserato anti-wtPLY antisera. Either or both of these assays may be utilizedto determine the activity of a particular mPLY. In vivo assays may alsobe used, including but not limited to the sepsis model, the focalpneumonia model, and the intranasal challenge models essentially asdescribed in Example 8. Thus, a suitable mPLY may be one that exhibitslower hemolytic activity than wtPLY (e.g., via an in vitro hemolysisassay). A suitable mPLY may also be one that, following administrationto a host, causes the host to produce antibodies that inhibit hemolysisby wtPLY (e.g., via a hemolysis inhibition assay), is immunogenic (e.g.,an immunogenic composition, one that induces the production ofantibodies or a cytotoxic T cell response against wtPLY ormicroorganisms expressing wtPLY), protective (e.g., a prophylactic ortherapeutic vaccine composition, one that induces an immune responsethat protects the host against infection by or limits analready-existing infection, respectively, by an organism expressingwtPLY), provides benefits as measured by any one or more of the in vivoassays described herein (e.g., sepsis model, focal pneumoniae model,intranasal challenge, lung damage assay), and/or increases or decreasesexpression of cytokines by macrophages as compared to wtPLY. It is to beunderstood that these methods for identifying and/or characterizing mPLYare exemplary and non-limiting; other assays may also be suitable.

In the in vitro hemolysis assay, test polypeptides (e.g., wtPLY, mPLY)may be separately serially diluted across a plastic microtiter plate inserial dilutions (e.g., 2-fold) with a highest concentration typicallybeing about 0.5 mg/mL of test polypeptide. Bovine serum albumin (BSA)may be included to prevent adsorptive losses on the plastic microtiterplate. Sheep red blood cells may then be added to all wells, and theplates incubated for a sufficient period of time (e.g., 30 min.).Positive controls (e.g., 100% lysis measurement is obtained by theaddition of 1% Trition X-100 to particular wells) and negative controls(e.g., PBS alone) are typically included. The plate may then becentrifuged to separate the intact cells from the lysed cells. Thesupernatant containing the lysed cells may then be transferred to afresh plate and subjected to an A₅₄₀ hemoglobin release assay (lysedcells release hemoglobin). The specific activity may be determined asthe inverse of protein concentration (mg/mL) at which 50% hemolysisoccurred relative to the positive control. Using this assay system, itis preferred that a particular mPLY have a specific activity of lessthan wtPLY. For example, mPLT may have a specific activity of about 0%,0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5% or <10% thespecific activity of wtPLY. A specific activity of approximately withinthese ranges typically indicates the particular mPLY is withinacceptable parameters.

In the hemolysis inhibition assay, anti-PLY (wtPLY or mPLY) antisera isproduced essentially as described in the art or herein and seriallydiluted (e.g., two-fold) across a microtiter plate (e.g., a 96-wellplate) and a constant amount of wtPLY is added to each well to beanalyzed. Sheep red blood cells are added to all wells and the assayproceeds as per the in vitro hemolysis assay described in the precedingparagraph. The data are plotted as percent hemolysis versus antiserumtiter (log scale). The 50% hemolysis inhibition titer is taken as theinverse of the serum dilution at which the percent hemolysis is reducedto 50%. Using this assay system, it is preferred that a particular mPLYhave a similar (e.g., anywhere from about 75 to about 100%) or higher50% hemolysis inhibition titer than wtPLY. For example, it is preferredthat a particular mPLY induce an antisera having a 50% hemolysisinhibition titer (using serial two-fold dilutions) of about 1:16, 1:32,1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048, 1:5096, or 1:10,192, orgreater. A 50% hemolysis inhibition titer of approximately within theseranges typically indicates the particular mPLY is within acceptableparameters.

In certain embodiments, it may be desired that the mPLY have both anacceptable specific activity as determined by the in vitro hemolysisassay and an acceptable 50% hemolysis inhibition titer. For instance, asuitable mPLY may have a specific activity (as determined using the invitro hemolysis assay) of about (referring to each of the followingvalues independently) 0%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%,0.5%, 1%, 5% or <10% the specific activity of wtPLY, and a 50% hemolysisinhibition titer (using serial two-fold dilutions) of about 1:128,1:256, 1:512, 1:1024, 1:2048, 1:5096, or 1:10,192.

wtPLY is known to activate macrophages to generate cytokines (Malley, etal. PNAS USA, 100(4):1966-71 (2003)). Suitable mPLY may be identified bydetermining macrophage activation by measuring mPly-induced cytokineproduction (e.g., (IL-1β, IL-6 and IL-10) in vitro. Macrophage-likecells (e.g., human MM6 and mouse J774A.1 cells) may be incubatedovernight with wtPLY or mPLY, and treated or not treated with proteinaseK and heat (to distinguish false positives due to lipopolysaccharide(LPS) contamination in the treated group). Cytokine production (e.g.,IL-6, IL-10, TNF-α, IL-1(3) may be measured by ELISA after overnightincubation. In certain embodiments, a suitable mPLY will induceexpression of cytokines (e.g., IL-1β, IL-6 and IL-10) more than willwtPLY. In other embodiments, a suitable mPLY will induce expression ofcytokines (e.g., IL-1β, IL-6 and IL-10) less than will wtPLY. Thedifference in the levels of expression induced by mPLY and wtPLY may be,for instance, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or morethan 100%.

It is also desired that antibodies generated against mPLY provide forlower levels of lung tissue damage induced by wtPLY or organismsexpressing wtPLY. To test the effectiveness of such antibodies usinganimal models, one or more mice (or other suitable model animal) may beimmunized with mPLY (e.g., a dose of about 1, 2, 5 or 10 μg for asufficient period of time), and then challenged with wtPLY (e.g., a doseof about 1, 2, 5, or 10 μg for a sufficient period of time) or anorganism expressing wtPLY. After a sufficient period of time (e.g., aperiod of hours, days, weeks), the lungs may be harvested and stained toobserve tissue damage. wtPLY typically causes perivascular edema,thickened, disrupted alveolar walls, diminished alveolar space, andfluid and blood infiltration of the alveolar spaces. In certainembodiments, a suitable mPLY may induce antibodies that reduce lungdamage by, for instance, about 10, 20, 30, 40, 50, 60, 70, 80, 90, or100% as compared to that induced by wtPLY or an organism expressingwtPLY.

In addition, in certain embodiments, it is preferred that a suitablemPLY exhibit immunogenic properties (e.g., inducing a neutralizingand/or protective immune response following administration to a host).The presence of neutralizing and/or protective immune response may bedemonstrated by demonstrating that infection by organisms expressingwtPLY is decreased in immunized individuals (e.g., human beings, orusing animal models). Suitable animal models include the sepsis model,the focal pneumoniae model, and the intranasal challenge model. In thesepsis model, test animals (e.g., mice or similar model) may beimmunized (e.g., subcutaneously, intravenously, intramuscularly,intradermally, intranodally, intranasally) with mPLY (with or withoutone or more adjuvants at suitable timepoints such as days 0, 7, 14, 21,28, 35, 42), and then after a suitable amount of time (e.g., about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 weeks) challenged by intranasal (IN)injection with a suitable amount of wt-PLY expressing organism (e.g.,10⁷ colony forming units (cfu) of S. pneumoniae serotype 6B). Followingthe challenge, mice may be monitored for mortality, and sample bleedsmay be drawn. Sera may be analyzed for total PLY-specific IgG responseusing, for example, an antibody ELISA and for PLY neutralizing capacityusing, for example, an inhibition of hemolysis assay. Statisticalanalysis (e.g., Fisher's exact test, Wilcoxon test, Mann-Whitney Test)may then be performed on the survival and ELISA/inhibition of hemolysisassay data to determine whether the mPLY is effective.

mPlyD1 may also be tested using a focal pneumonia mouse model. Briefly,animals may be immunized (e.g., subcutaneously, intravenously,intramuscularly, intradermally, intranodally, intranasally) with apurified recombinant PlyD1 protein with or without adjuvant. Testanimals (e.g., mice or similar model) may be immunized with mPLY (withor without one or more adjuvants at suitable timepoints such as days 0,7, 14, 21, 28, 35, 42), and then after a suitable amount of time (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks) challenged by intranasally(IN) with a suitable amount of wt-PLY expressing organism (e.g., 3-7×10⁶cfu of S. pneumoniae strain EF3030 (serotype 19F)). Following thechallenge, mice may be monitored for mortality, and sample bleeds may bedrawn. Sera may be analyzed for total PLY-specific IgG response using,for example, an antibody ELISA and for PLY neutralizing capacity using,for example, an inhibition of hemolysis assay. Statistical analysis(e.g., Fisher's exact test, Wilcoxon test, Mann-Whitney Test) may thenbe performed on the survival and ELISA/inhibition of hemolysis assaydata to determine whether the mPLY is effective.

The effectiveness of mPlyD1 as a vaccine may also be evaluated in-houseusing an intranasal challenge mouse model. In this model, mice may beimmunized (e.g., subcutaneously, intravenously, intramuscularly,intradermally, intranodally, intranasally) with purified recombinantPlyD1 proteins at an effective dose (e.g., from about 0.25 to 25 μg)with or without one or more adjuvants at suitable timepoints such asdays 0, 7, 14, 21, 28, 35, 42), and then after a suitable amount of time(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks) challenged byintranasally (IN) with a suitable amount of wt-PLY expressing organism(e.g., a lethal dose (5×10⁵ cfu) of S. pneumoniae strain 14453 (serotype6B)). Following the challenge, mice may be monitored for mortality, andsample bleeds may be drawn. Sera may be analyzed for total PLY-specificIgG response using, for example, an antibody ELISA and for PLYneutralizing capacity using, for example, an inhibition of hemolysisassay. Statistical analysis (e.g., Fisher's exact test, Wilcoxon test,Mann-Whitney Test) may then be performed on the survival andELISA/inhibition of hemolysis assay data to determine whether the mPLYis effective.

Immunogenic compositions and vaccines containing mPLY polypeptidesand/or fragments thereof may be used to treat diseases such as, forexample, invasive pneumococcal such as pneumonia, meningitis, otitismedia, and bacteremia. The polypeptides and fragments exhibitsubstantially reduced toxicity compared to the native toxin. Nucleicacid sequences encoding the mPLY polypeptides and/or fragments, vectorscontaining such nucleic acid sequences, and host cells capable ofexpressing the mutant PLY polypeptides and/or fragments are alsoprovided. The mPLY polypeptides and/or fragments preferably exhibitdecreased toxicity relative to wtPLY (e.g., lower hemolytic/cytolyticactivity) and elicit neutralizing antibodies which are cross-reactivewith antibodies elicited by the wtPLY. mPLY polypeptides and/orfragments may be used to produce immunological compositions which mayinclude vaccines. An immunological composition is one that, uponadministration to a host (e.g., an animal), induces or enhances animmune response directed against the antigen or immunogen containedwithin the composition. This response may include the generation ofantibodies (e.g, through the stimulation of B cells) or a T cell-basedresponse (e.g., a cytolytic response). These responses may or may not beprotective or neutralizing. A protective or neutralizing immune responseis one that is detrimental to the infectious organism corresponding tothe antigen (e.g, from which the antigen was derived) and beneficial tothe host (e.g., by reducing or preventing infection). As used herein,protective or neutralizing antibodies and/or cellular responses may bereactive to mPLY, wtPLY, or fragments thereof and reduce or inhibit thelethality of wtPLY (or organisms expressing wtPLY or mPLY) when testedin animals. An immunological composition that, upon administration to ahost, results in a protective or neutralizing immune response, may beconsidered a vaccine. Immunological compositions comprising one or moremPLY polypeptides may also include one or more additional antigens, suchas one or more antigens of S. pneumoniae. Exemplary antigens include,for example, PcPA and/or PhtD. Other variations of immunologicalcompositions are also contemplated as would be understood by one ofskill in the art.

As mentioned above, mPLY polypeptides and/or fragments thereof typicallyhave at least one nucleic acid and/or one amino acid substitution.Modified polypeptides may also exhibit at least one change in abiological function (e.g., immunogenicity, haemolytic activity) comparedwith a wtPLY. mPLY polypeptides or fragments thereof are preferablysubstantially nontoxic when compared to a wtPLY protein at the samedose, elicit antibodies that are preferably protective or neutralizingand cross-reactive with antibodies elicited by a wtPLY protein. A mPLYpolypeptide and/or fragment thereof may be generated using a variety ofmethods including, but not limited to, site-directed mutagenesis, randommutagenesis, conventional mutagenesis, in vitro mutagenesis, spontaneousmutagenesis and chemical synthesis. Methods of mutagenesis can be foundin Sambrook et al., A Guide to Molecular Cloning, Cold Spring Harbour,N.Y. (1989) and Sambrook and Russel. Molecular Cloning: A LaboratoryManual (2001), for instance.

This disclosure further relates to antibodies, preferably protective orneutralizing antibodies, generated using a mPLY polypeptide or fragmentthereof where the resultant antibodies are reactive to wtPLY and/orfragments thereof. Also provided are methods for eliciting theproduction of antibodies, which may be protective and/or neutralizing,reactive to the mPLY or fragments thereof. The antibodies may elicitboth active and passive immunity. The mPLY polypeptides and/or fragmentsthereof may also be used to identify and isolate antibodies, which maybe protective and/or neutralizing, that are cross-reactive with thoseelicited by wtPLY.

Nucleic acids encoding mPLY polypeptides are also provided. Alsoprovided are variants of such sequences, including degenerate variantsthereof. In certain embodiments, a nucleic acid molecule encoding themPLY polypeptide and/or fragment may be inserted into one or moreexpression vectors, as discussed below in greater detail. In suchembodiments, the mPLY polypeptide and/or fragment is/are encoded bynucleotides corresponding to the amino acid sequence. The particularcombinations of nucleotides that encode the various amino acids are wellknown in the art, as described in various references used by thoseskilled in the art (i.e., Lewin, B. Genes V, Oxford University Press,1994), and as shown in Table 1 below. Nucleic acid variants may use anycombination of nucleotides that encode the polypeptide of interest.

TABLE 1 Phe (F) TTT Ser (S) TCT Tyr (Y) TAT Cys (C) TGT TTC TCC TAC TGCLeu (L) TTA TCA TERM TAA TERM TGA TTG TCG TAG Trp (W) TGG CTT Pro (P)CCT His (H) CAT Arg (R) CGT CTC CCC CAC CGC CTA CCA Gln (Q) CAA CGA CTGCCG CAG CGG Ile (I) ATT Thr (T) ACT Asn (N) AAT Ser (S) AGT ATC ACC AACAGC ATA ACA Lys (K) AAA Arg (R) AGA Met (M) ATG ACG AAG AGG Val (V) GTTAla (A) GCT Asp (D) GAT Gly (G) GGT GTC GCC GAC GGC GTA GCA  Glu (E) GAAGGA GTG GCG GAG GGG

Modified PLY polypeptides and/or fragments are typically selected toinclude least one amino acid substitution, and optionally at least onechange in a biological function compared with wtPLY. Modified PLYproteins or fragment thereof may be selected to ensure that they aresubstantially non-toxic when compared to a protein substantiallycorresponding to a wtPLY protein at the same dose and elicitneutralizing antibodies which may be cross-reactive with antibodieselicited by a wtPLY protein. As described herein, mPLY polypeptides andfragments thereof may be useful in immunogenic compositions or vaccinesagainst invasive pneumococcal disease (e.g., pneumonia, meningitis,otitis media, bacteremia) caused by Streptococcus pneumoniae.

Amino acid substitution may be conservative or non-conservative.Substitutions that result in a structural change that may affect mPLYactivity include the following:

-   -   1. change from one type of charge to another (e.g., arginine to        glutamate);    -   2. change from charge to noncharged (e.g., glutamate to        proline);    -   3. change in cysteine residues and formation of disulfide bonds        (e.g., glutamate to cysteine and threonine to cysteine);    -   4. change from hydrophobic to hydrophilic residues (e.g.,        alanine to serine);    -   5. change from hydrophilic residues to hydrophobic residues        (e.g., aspartate to leucine);    -   6. change in size of the amino acid (e.g., glycine to valine);    -   7. change to a conformationally restrictive amino acid or analog        (e.g., glutamate to proline); and    -   8. change to a non-naturally occurring amino acid or analog.

Conservative amino acid substitutions may involve a substitution of anative amino acid residue with a non-native residue such that there islittle or no effect on the size, polarity, charge, hydrophobicity, orhydrophilicity of the amino acid residue at that position and, inparticular, does not result in decreased immunogenicity. Suitableconservative amino acid substitutions are shown in Table 2.

TABLE 2 Preferred Original Conservative Residues Exemplary ConservativeSubstitutions Substitution Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp GlyPro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe,Norleucine Leu Leu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4Diamino-butyric Acid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val,Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr,Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala,Norleucine LeuThe specific amino acid substitution selected may depend on the locationof the site selected.

In certain embodiments, nucleic acids encoding mPLY polypeptides and/orfragments substituted may be selected based on the degeneracy of thegenetic code. Where the nucleic acid is a recombinant DNA moleculeuseful for expressing a polypeptide in a cell (e.g., an expressionvector), such a substation may result in the expression of a polypeptidewith the same amino acid sequence as that originally encoded by the DNAmolecule. As described above, however, substitutions may beconservative, or non-conservative, or any combination thereof.

A skilled artisan will be able to determine suitable variants of mPLYpolypeptides and/or fragments provided herein using well-knowntechniques. For identifying suitable areas of the molecule that may bechanged without destroying biological activity (i.e., pore-formingactivity, red blood cell (RBC) agglutination, RBC hemolysis, MHCbinding, immunogenicity), one skilled in the art may target areas notbelieved to be important for that activity. For example, when PLYderivatives with similar activities from the same species or from otherspecies are known, one skilled in the art may compare the amino acidsequence of a polypeptide to such similar polypeptides. By performingsuch analyses, one can identify residues and portions of the moleculesthat are conserved. It will be appreciated that changes in areas of themolecule that are not conserved relative to such similar pneumolysinderivatives would be less likely to adversely affect the biologicalactivity and/or structure of a polypeptide. Similarly, the residuesrequired for binding to MHC are known, and may be mutated to improvebinding of PLY antigenic sequences to MHC molecules. However,modifications resulting in decreased binding to MHC will not beappropriate in most situations. One skilled in the art would also knowthat, even in relatively conserved regions, one may substitutechemically similar amino acids for the naturally occurring residueswhile retaining the desired characteristics of the polypeptide and/orfragment. Therefore, even areas that may be important for biologicalactivity or for structure may be subject to conservative amino acidsubstitutions without destroying the biological activity or withoutadversely affecting the structure of the mPLY derivative.

In other embodiments, the mPLY polypeptides and/or fragments describedherein may include fusion polypeptide segments that assist inpurification or detection of the polypeptides. Fusions can be madeeither at the amino terminus or at the carboxy terminus of the subjectpolypeptide variant thereof. Fusions may be direct with no linker oradapter molecule or may be through a linker or adapter molecule. Alinker or adapter molecule may be one or more amino acid residues,typically from about 2 to about 50 amino acid residues. A linker oradapter molecule may also be designed with a cleavage site for a DNArestriction endonuclease or for a protease to allow for the separationof the fused moieties. It will be appreciated that once constructed, thefusion polypeptides can be derivatized according to the methodsdescribed herein. Suitable fusion segments include, among others, metalbinding domains (e.g., a poly-histidine segment), immunoglobulin bindingdomains (i.e., Protein A, Protein G, T cell, B cell, Fc receptor, orcomplement protein antibody-binding domains), sugar binding domains(e.g., a maltose binding domain), and/or a “tag” domain (i.e., at leasta portion of α-galactosidase, a strepavidin-derived tag peptide, a T7tag peptide, a FLAG peptide, or other peptides that can be purifiedusing compounds that bind to the domain, such as monoclonal antibodies).This tag is typically fused to the polypeptide upon expression of thepolypeptide, and can serve as a means for affinity purification of thesequence of interest polypeptide from the host cell. Affinitypurification can be accomplished, for example, by column chromatographyusing antibodies against the tag as an affinity matrix. Optionally, thetag can subsequently be removed from the purified sequence of interestpolypeptide by various means such as using certain peptidases forcleavage.

In certain embodiments, the mPLY polypeptides and/or fragments may bedirectly or indirectly (i.e., using an antibody) labeled or tagged in amanner which enables it to be detected. Labels include fluorochromessuch as fluorescein, rhodamine, phycoerythrin, Europium and Texas Red,chromogenic dyes such as diaminobenzidine, radioisotopes, macromolecularcolloidal particles or particulate material such as latex beads that arecoloured, magnetic or paramagnetic, binding agents such as biotin anddigoxigenin, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded, for example in a FACS,ELISA, Western blot, TRFIA, immunohistochemistry, evanescence, Luminexbead array, or dipstick or other lateral flow assay format. Suitableantibody-binding molecules for use in such methods may includeimmunoglobulin-binding antibodies, for example anti-human Ig antibodies,anti-human Ig antibodies, anti-human antibodies specific for Ig isotypesor for subclasses of IgG, or specific for Staphylococcal protein A or G.

Preferred fluorescent tag proteins include those derived from the jellyfish protein known as green fluorescent protein (GFP). Furtherinformation on GFP and other fluorophores is given in the followingpublications: Tsien R Y, “The Green Fluorescent Protein” Annual Reviewsof Biochemistry 1998; 67:509-544 Verkhusha, V and Lukyanov, K. “TheMolecular Properties and Applications of Anthoza Fluorescent Proteinsand Chromophores” Nature Biotechnology 2004; 22:289-296. Plasmid vectorsencoding a wide range of fluorescent tag proteins are commerciallyavailable from various suppliers including an array of “LivingColours&#8482; Fluorescent Proteins” available commercially fromClontech Laboratories, Inc. Similar vectors can also be obtained fromother suppliers including Invitrogen and Amersham Biosciences. Suitablefluorescent proteins derived from GFP are the red-shifted variant EGFP,the cyan shifted variant ECFP and the yellow shifted variant EYFP. EGFPis preferred as the fluorescent marker because it gives brightfluorescence combined with minimal effect on the antigenic properties ofthe target antigen. Alternative fluorescent marker proteins arecommercially available. Biologically or chemically active agents includeenzymes, which catalyse reactions that develop or change colours orcause changes in electrical properties, for example, and may also beutilized. They may be molecularly excitable, such that electronictransitions between energy states result in characteristic spectralabsorptions or emissions. They may include chemical entities used inconjunction with biosensors. Biotin/avidin or biotin/streptavidin andalkaline phosphatase detection systems may be employed. Further examplesinclude horseradish peroxidase and chemiluminescence In someembodiments, the non-immobilized, antibody-binding molecule, orpolypeptide may be detected using an antibody which binds to saidnon-immobilised antibody-binding molecule or polypeptide. A suitabledetection antibody may be labeled by means of fluorescence. The labelmay be a fluorescent marker (tag) which is used to label the targetantigen directly such that the antigen and the fluorescent marker form afusion protein.

If antibodies against the target antigen are present in a biologicalsample, the antigen may be labeled with the tag bound to thoseantibodies, and the complex formed thereby detected usingimmunoprecipitation. The fluorescence associated with the tag may thenbe used to determine that protein has been precipitated (qualitativedetermination) or to determine the amount of protein precipitated(quantitative determination). For example, soluble extracts of afluorescence-tagged antigen may be incubated with patient sera for anappropriate period of time such as overnight at 4° C. (typically 10-15μl of serum to 300-500 μl of extract or less) to allow antibodies tobind to the antigen. Protein A or Protein G Sepharose beads,preincubated with low IgG fetal calf serum (Sigma) to block non-specificbinding, are then added to the extract/serum mix containing the taggedprotein/antibody complexes, and mixed with gentle rotation for 1 to 2hours at room temperature. The antibodies within the serum, includingthose that specifically bind the tagged protein, are bound by theprotein A/G beads. The protein A/G Sepharose beads are then washed in asuitable buffer (typically 10 mM Tris-HCl pH 7.4, 100 mM NaCl/ImMEDTA/1% Triton X-100) to remove any unbound tagged protein. This may beachieved by three rounds of centrifugation, removal of the supernatant,and resuspension in buffer. The beads, some with tagged proteinattached, are then collected and placed in a fluorescence reader, forexample a Spectra Max Gemini XS plate reader from Molecular Devices Inc.The presence of specific autoantibodies/antibodies in the original serumsample is quantitated. In the case of GFP this uses excitation atwavelength 472 nm and emission at 512 nm. The fluorescence excitationwill depend upon the fluorophore/tag that is used but it would bepossible to combine several different tagged proteins in the same time.For example, different mutant PLY polypeptides and/or fragments thereofmay be separately tagged and separately or simultaneously assayed. Thesensitivity of the method is dependent on the detection device and canbe considerably enhanced by using more sensitive detection devices.Various modifications of these methods could also be utilized.

The assays described herein for detecting antibodies immunoreactive withstreptococcal antigens may also be combined with other assays useful fordetecting streptococcal infection. For instance, these assays (i.e.,ELISA) may be combined with polymerase chain reaction (PCR) assays fordetecting streptococcal nucleic acid in a biological sample.Alternatively, an ELISA assay may be combined with animmunoprecipitation assay. Or, a PCR-based assay may be combined with animmunoprecipitation assay. Combining the various assays described hereinmay serve to even further increase the sensitivity of detection andfurther decrease the negative predictive value of the data.

As previously mentioned, expression vectors may also be suitable foruse. Expression vectors are typically comprised of a flanking sequenceoperably linked to a heterologous nucleic acid sequence encoding apolypeptide (the “coding sequence”). In other embodiments, or incombination with such embodiments, a flanking sequence is preferablycapable of effecting the replication, transcription and/or translationof the coding sequence and is operably linked to a coding sequence. Tobe “operably linked” indicates that the nucleic acid sequences areconfigured so as to perform their usual function. For example, apromoter is operably linked to a coding sequence when the promoter iscapable of directing transcription of that coding sequence. A flankingsequence need not be contiguous with the coding sequence, so long as itfunctions correctly. Thus, for example, intervening untranslated yettranscribed sequences can be present between a promoter sequence and thecoding sequence and the promoter sequence can still be consideredoperably linked to the coding sequence. Flanking sequences may behomologous (i.e., from the same species and/or strain as the host cell),heterologous (i.e., from a species other than the host cell species orstrain), hybrid (i.e., a combination of flanking sequences from morethan one source), or synthetic. A flanking sequence may also be asequence that normally functions to regulate expression of thenucleotide sequence encoding the polypeptide in the genome of the hostmay also be utilized.

In certain embodiments, it is preferred that the flanking sequence is atranscriptional regulatory region that drives high-level gene expressionin the target cell. The transcriptional regulatory region may comprise,for example, a promoter, enhancer, silencer, repressor element, orcombinations thereof. The transcriptional regulatory region may beeither constitutive or tissue- or cell-type specific (i.e., the regiondrives higher levels of transcription in one type of tissue or cell ascompared to another). As such, the source of a transcriptionalregulatory region may be any prokaryotic or eukaryotic organism, anyvertebrate or invertebrate organism, or any plant, provided that theflanking sequence is functional in, and can be activated by, the hostcell machinery. A wide variety of transcriptional regulatory regions maybe utilized.

Suitable transcriptional regulatory regions include, among others, theCMV promoter (i.e., the CMV-immediate early promoter); promoters fromeukaryotic genes (i.e., the estrogen-inducible chicken ovalbumin gene,the interferon genes, the gluco-corticoid-inducible tyrosineaminotransferase gene, and the thymidine kinase gene); and the majorearly and late adenovirus gene promoters; the SV40 early promoter region(Bernoist and Chambon, 1981, Nature 290:304-10); the promoter containedin the 3′ long terminal repeat (LTR) of Rous sarcoma virus (RSV)(Yamamoto, et al., 1980, Cell 22:787-97); the herpes simplex virusthymidine kinase (HSV-TK) promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:1444-45); the regulatory sequences of themetallothionine gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.,75:3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad.Sci. U.S.A., 80:21-25). Tissue- and/or cell-type specifictranscriptional control regions include, for example, the elastase Igene control region which is active in pancreatic acinar cells (Swift etal., 1984, Cell 38:639-46; Ornitz et al., 1986, Cold Spring Harbor Symp.Quant. Biol. 50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-515);the insulin gene control region which is active in pancreatic beta cells(Hanahan, 1985, Nature 315:115-22); the immunoglobulin gene controlregion which is active in lymphoid cells (Grosschedl et al., 1984, Cell38:647-58; Adames et al., 1985, Nature 318:533-38; Alexander et al.,1987, Mol. Cell. Biol., 7:1436-44); the mouse mammary tumor viruscontrol region in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-95); the albumin gene control region in liver(Pinkert et al., 1987, Genes and Devel. 1:268-76); thealpha-feto-protein gene control region in liver (Krumlauf et al., 1985,Mol. Cell. Biol., 5:1639-48; Hammer et al., 1987, Science 235:53-58);the alpha 1-antitrypsin gene control region in liver (Kelsey et al.,1987, Genes and Devel. 1:161-71); the beta-globin gene control region inmyeloid cells (Mogram et al., 1985, Nature 315:338-40; Kollias et al.,1986, Cell 46:89-94); the myelin basic protein gene control region inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-12); the myosin light chain-2 gene control region in skeletalmuscle (Sani, 1985, Nature 314:283-86); and the gonadotropic releasinghormone gene control region in the hypothalamus (Mason et al., 1986,Science 234:1372-78), and the tyrosinase promoter in melanoma cells(Hart, I. Semin Oncol 1996 February; 23(1):154-8; Siders, et al. CancerGene Ther 1998 September-October; 5(5):281-91). Other suitable promotersare known in the art.

The nucleic acid molecule may be administered as part of a viral andnon-viral vector. In one embodiment, a DNA vector is utilized to delivernucleic acids encoding the targeted immunogen and/or associatedmolecules (i.e., co-stimulatory molecules, cytokines or chemokines) tothe patient. In doing so, various strategies may be utilized to improvethe efficiency of such mechanisms including, for example, the use ofself-replicating viral replicons (Caley, et al. 1999. Vaccine, 17:3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al.2000. Cancer Res. 60: 51-55), codon optimization (Liu, et al. 2000. Mol.Ther., 1: 497-500; Dubensky, supra; Huang, et al. 2001. J. Virol. 75:4947-4951), in vivo electroporation (Widera, et al. 2000. J. Immunol.164: 4635-3640), incorporation of nucleic acids encoding co-stimulatorymolecules, cytokines and/or chemokines (Xiang, et al. 1995. Immunity, 2:129-135; Kim, et al. 1998. Eur. J. Immunol., 28: 1089-1103; Iwasaki, etal. 1997. J. Immunol. 158: 4591-3601; Sheerlinck, et al. 2001. Vaccine,19: 2647-2656), incorporation of stimulatory motifs such as CpG(Gurunathan, supra; Leitner, supra), sequences for targeting of theendocytic or ubiquitin-processing pathways (Thomson, et al. 1998. J.Virol. 72: 2246-2252; Velders, et al. 2001. J. Immunol. 166: 5366-5373),prime-boost regimens (Gurunathan, supra; Sullivan, et al. 2000. Nature,408: 605-609; Hanke, et al. 1998. Vaccine, 16: 439-445; Amara, et al.2001. Science, 292: 69-74), proteasome-sensitive cleavage sites, and theuse of mucosal delivery vectors such as Salmonella (Darji, et al. 1997.Cell, 91: 765-775; Woo, et al. 2001. Vaccine, 19: 2945-2954). Othermethods are known in the art, some of which are described below.

Various viral vectors that have been successfully utilized forintroducing a nucleic acid to a host include retrovirus, adenovirus,adeno-associated virus (AAV), herpes virus, and poxvirus, among others.The vectors may be constructed using standard recombinant techniqueswidely available to one skilled in the art. Such techniques may be foundin common molecular biology references such as Molecular Cloning: ALaboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor LaboratoryPress), Gene Expression Technology (Methods in Enzymology, Vol. 185,edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), and PCRProtocols: A Guide to Methods and Applications (Innis, et al. 1990.Academic Press, San Diego, Calif.).

Preferred retroviral vectors are derivatives of lentivirus as well asderivatives of murine or avian retroviruses. Examples of suitableretroviral vectors include, for example, Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A number ofretroviral vectors can incorporate multiple exogenous nucleic acidsequences. As recombinant retroviruses are defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided by, for example, helper cell lines encodingretrovirus structural genes. Suitable helper cell lines include Ψ2,PA317 and PA12, among others. The vector virions produced using suchcell lines may then be used to infect a tissue cell line, such as NIH3T3 cells, to produce large quantities of chimeric retroviral virions.Retroviral vectors may be administered by traditional methods (i.e.,injection) or by implantation of a “producer cell line” in proximity tothe target cell population (Culver, K., et al., 1994, Hum. Gene Ther., 5(3): 343-79; Culver, K., et al., Cold Spring Harb. Symp. Quant. Biol.,59: 685-90); Oldfield, E., 1993, Hum. Gene Ther., 4 (1): 39-69). Theproducer cell line is engineered to produce a viral vector and releasesviral particles in the vicinity of the target cell. A portion of thereleased viral particles contact the target cells and infect thosecells, thus delivering a nucleic acid to the target cell. Followinginfection of the target cell, expression of the nucleic acid of thevector occurs.

Adenoviral vectors have proven especially useful for gene transfer intoeukaryotic cells (Rosenfeld, M., et al., 1991, Science, 252 (5004):431-3; Crystal, R., et al., 1994, Nat. Genet., 8 (1): 42-51), the studyof eukaryotic gene expression (Levrero, M., et al., 1991, Gene, 101 (2):195-202), vaccine development (Graham, F. and Prevec, L., 1992,Biotechnology, 20: 363-90), and in animal models (Stratford-Perricaudet,L., et al., 1992, Bone Marrow Transplant., 9 (Suppl. 1): 151-2; Rich,D., et al., 1993, Hum. Gene Ther., 4 (4): 461-76). Experimental routesfor administering recombinant Ad to different tissues in vivo haveincluded intratracheal instillation (Rosenfeld, M., et al., 1992, Cell,68 (1): 143-55) injection into muscle (Quantin, B., et al., 1992, Proc.Natl. Acad. Sci. U.S.A., 89 (7): 2581-3), peripheral intravenousinjection (Herz, J., and Gerard, R., 1993, Proc. Natl. Acad. Sci.U.S.A., 90 (7): 2812-6) and stereotactic inoculation to brain (Le Gal LaSalle, G., et al., 1993, Science, 259 (5097): 988-90), among others.

Adeno-associated virus (AAV) demonstrates high-level infectivity, broadhost range and specificity in integrating into the host cell genome(Hermonat, P., et al., 1984, Proc. Natl. Acad. Sci. U.S.A., 81 (20):6466-70). And Herpes Simplex Virus type-1 (HSV-1) is yet anotherattractive vector system, especially for use in the nervous systembecause of its neurotropic property (Geller, A., et al., 1991, TrendsNeurosci., 14 (10): 428-32; Glorioso, et al., 1995, Mol. Biotechnol., 4(1): 87-99; Glorioso, et al., 1995, Annu. Rev. Microbiol., 49: 675-710).

Poxvirus is another useful expression vector (Smith, et al. 1983, Gene,25 (1): 21-8; Moss, et al, 1992, Biotechnology, 20: 345-62; Moss, et al,1992, Curr. Top. Microbiol. Immunol., 158: 25-38; Moss, et al. 1991.Science, 252: 1662-1667). Poxviruses shown to be useful includevaccinia, NYVAC, avipox, fowlpox, canarypox, ALVAC, and ALVAC(2), amongothers.

NYVAC (vP866) was derived from the Copenhagen vaccine strain of vacciniavirus by deleting six nonessential regions of the genome encoding knownor potential virulence factors (see, for example, U.S. Pat. Nos.5,364,773 and 5,494,807). The deletion loci were also engineered asrecipient loci for the insertion of foreign genes. The deleted regionsare: thymidine kinase gene (TK; J2R) vP410; hemorrhagic region (u;B13R+B14R) vP553; A type inclusion body region (ATI; A26L) vP618;hemagglutinin gene (HA; A56R) vP723; host range gene region (C7L-K1L)vP804; and, large subunit, ribonucleotide reductase (I4L) vP866. NYVACis a genetically engineered vaccinia virus strain that was generated bythe specific deletion of eighteen open reading frames encoding geneproducts associated with virulence and host range. NYVAC has been showto be useful for expressing TAs (see, for example, U.S. Pat. No.6,265,189). NYVAC (vP866), vP994, vCP205, vCP1433, placZH6H4Lreverse,pMPC6H6K3E3 and pC3H6FHVB were also deposited with the ATCC under theterms of the Budapest Treaty, accession numbers VR-2559, VR-2558,VR-2557, VR-2556, ATCC-97913, ATCC-97912, and ATCC-97914, respectively.

ALVAC-based recombinant viruses (i.e., ALVAC-1 and ALVAC-2) are alsosuitable for use (see, for example, U.S. Pat. No. 5,756,103). ALVAC(2)is identical to ALVAC(1) except that ALVAC(2) genome comprises thevaccinia E3L and K3L genes under the control of vaccinia promoters (U.S.Pat. No. 6,130,066; Beattie et al., 1995a, 1995b, 1991; Chang et al.,1992; Davies et al., 1993). Both ALVAC(1) and ALVAC(2) have beendemonstrated to be useful in expressing foreign DNA sequences, such asTAs (Tartaglia et al., 1993 a,b; U.S. Pat. No. 5,833,975). ALVAC wasdeposited under the terms of the Budapest Treaty with the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209, USA, ATCC accession number VR-2547.

Another useful poxvirus vector is TROVAC. TROVAC refers to an attenuatedfowlpox that was a plaque-cloned isolate derived from the FP-1 vaccinestrain of fowlpoxvirus which is licensed for vaccination of 1 day oldchicks. TROVAC was likewise deposited under the terms of the BudapestTreaty with the ATCC, accession number 2553.

“Non-viral” plasmid vectors may also be suitable in certain embodiments.Preferred plasmid vectors are compatible with bacterial, insect, and/ormammalian host cells. Such vectors include, for example, PCR-II, pCR3,and pcDNA3.1 (Invitrogen, San Diego, Calif.), pBSII (Stratagene, LaJolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL(BlueBacII, Invitrogen), pDSR-alpha (PCT pub. No. WO 90/14363) andpFastBacDual (Gibco-BRL, Grand Island, N.Y.) as well as Bluescript®plasmid derivatives (a high copy number COLE1-based phagemid, StratageneCloning Systems, La Jolla, Calif.), PCR cloning plasmids designed forcloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® kit, PCR2.1®plasmid derivatives, Invitrogen, Carlsbad, Calif.). Bacterial vectorsmay also be used. These vectors include, for example, Shigella,Salmonella, Vibrio cholerae, Lactobacillus, Bacille calmette guerin(BCG), and Streptococcus (see for example, WO 88/6626; WO 90/0594; WO91/13157; WO 92/1796; and WO 92/21376). Many other non-viral plasmidexpression vectors and systems are known in the art and may be used.

Other delivery techniques may also suffice including, for example,DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injectionof DNA, CaPO₄ precipitation, gene gun techniques, electroporation, andcolloidal dispersion systems. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system is a liposome,which are artificial membrane vesicles useful as delivery vehicles invitro and in vivo. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form (Fraley, R., et al., 1981, Trends Biochem. Sci., 6: 77). Thecomposition of the liposome is usually a combination of phospholipids,particularly high-phase-transition-temperature phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations. Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

A cultured cell comprising the vector is also provided. The culturedcell may be a cultured cell transfected with the vector or a progeny ofthe cell, wherein the cell expresses the immunogenic polypeptide.Suitable cell lines are known to those of skill in the art and arecommercially available, for example, through the American Type CultureCollection (ATCC). The transfected cells can be used in a method ofproducing an immunogenic polypeptide. The method comprises culturing acell comprising the vector under conditions that allow expression of theimmunogenic polypeptide, optionally under the control of an expressionsequence. The immunogenic polypeptide can be isolated from the cell orthe culture medium using standard protein purification methods.

The polypeptides and nucleic acids described herein may be combined withone or more pharmaceutically acceptable carriers prior to administrationto a host. A pharmaceutically acceptable carrier is a material that isnot biologically or otherwise undesirable, i.e., the material may beadministered to a subject, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

Suitable pharmaceutical carriers and their formulations are describedin, for example, Remington's: The Science and Practice of Pharmacy,21^(st) Edition, David B. Troy, ed., Lippicott Williams & Wilkins(2005). Typically, an appropriate amount of apharmaceutically-acceptable salt is used in the formulation to renderthe formulation isotonic. Examples of the pharmaceutically-acceptablecarriers include, but are not limited to, sterile water, saline,buffered solutions like Ringer's solution, and dextrose solution. The pHof the solution is generally from about 5 to about 8 or from about 7 toabout 7.5. Other carriers include sustained-release preparations such assemipermeable matrices of solid hydrophobic polymers containingpolypeptides or fragments thereof. Matrices may be in the form of shapedarticles, e.g., films, liposomes or microparticles. It will be apparentto those persons skilled in the art that certain carriers may be morepreferable depending upon, for instance, the route of administration andconcentration of composition being administered. Carriers are thosesuitable for administration of polypeptides and/or fragments thereof tohumans or other subjects.

Pharmaceutical compositions may also include carriers, thickeners,diluents, buffers, preservatives, surface active agents, adjuvants,immunostimulants, in addition to the immunogenic polypeptide.Pharmaceutical compositions may also include one or more activeingredients such as antimicrobial agents, antiinflammatory agents andanesthetics. Adjuvants may also be included to stimulate or enhance theimmune response. Non-limiting examples of suitable classes of adjuvantsinclude those of the gel-type (i.e., aluminum hydroxide/phosphate (“alumadjuvants”), calcium phosphate, microbial origin (muramyl dipeptide(MDP)), bacterial exotoxins (cholera toxin (CT), native cholera toxinsubunit B (CTB), E. coli labile toxin (LT), pertussis toxin (PT), CpGoligonucleotides, BCG sequences, tetanus toxoid, monophosphoryl lipid A(MPL) of, for example, E. coli, Salmonella minnesota, Salmonellatyphimurium, or Shigella exseri), particulate adjuvants (biodegradable,polymer microspheres), immunostimulatory complexes (ISCOMs)),oil-emulsion and surfactant-based adjuvants (Freund's incompleteadjuvant (FIA), microfluidized emulsions (MF59, SAF), saponins (QS-21)),synthetic (muramyl peptide derivatives (murabutide, threony-MDP),nonionic block copolymers (L121), polyphosphazene (PCCP), syntheticpolynucleotides (poly A:U, poly I:C), thalidomide derivatives(CC-4407/ACTIMID)), RH3-ligand, or polylactide glycolide (PLGA)microspheres, among others. Fragments, homologs, derivatives, andfusions to any of these toxins are also suitable, provided that theyretain adjuvant activity. Suitable mutants or variants of adjuvants aredescribed, e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627(Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PTmutant). Additional LT mutants that may used include, for example,Ser-63-Lys, Ala-69-Gly,Glu-110-Asp, and Glu-112-Asp mutants.

Metallic salt adjuvants such as alum adjuvants are well-known in the artas providing a safe excipient with adjuvant activity. The mechanism ofaction of these adjuvants are thought to include the formation of anantigen depot such that antigen may stay at the site of injection for upto 3 weeks after administration, and also the formation ofantigen/metallic salt complexes which are more easily taken up byantigen presenting cells. In addition to aluminium, other metallic saltshave been used to adsorb antigens, including salts of zinc, calcium,cerium, chromium, iron, and berilium. The hydroxide and phosphate saltsof aluminium are the most common. Formulations or compositionscontaining aluminium salts, antigen, and an additional immunostimulantare known in the art. An example of an immunostimulant is3-de-O-acylated monophosphoryl lipid A (3D-MPL).

In one embodiment of adjuvanted immunization, for example, mPLYpolypeptides and/or fragments thereof may be covalently coupled tobacterial polysaccharides to form polysaccharide conjugates. Suchconjugates may be useful, for example, as immunogens for eliciting a Tcell dependent immunogenic response directed against the bacterialpolysaccharide conjugated to the mPLY and/or fragments thereof.

One or more cytokines may also be suitable co-stimulatory components foruse with the mPLY polypeptides and/or fragments thereof, either aspolypeptides or as encoded by nucleic acids (Parmiani, et al. ImmunolLett 2000 Sep. 15; 74(1): 41-3; Berzofsky, et al. Nature Immunol. 1:209-219). Suitable cytokines include, for example, interleukin-2 (IL-2)(Rosenberg, et al. Nature Med. 4: 321-327 (1998)), IL-4, IL-7, IL-12(reviewed by Pardoll, 1992; Harries, et al. J. Gene Med. 2000July-August; 2(4):243-9; Rao, et al. J. Immunol. 156: 3357-3365 (1996)),IL-15 (Xin, et al. Vaccine, 17:858-866, 1999), IL-16 (Cruikshank, et al.J. Leuk Biol. 67(6): 757-66, 2000), IL-18 (J. Cancer Res. Clin. Oncol.2001. 127(12): 718-726), GM-CSF (CSF (Disis, et al. Blood, 88: 202-210(1996)), tumor necrosis factor-alpha (TNF-α), or interferon-gamma(INF-γ). Other cytokines may also be suitable for use, as is known inthe art.

The term “antibody” or “antibodies” includes whole or fragmentedantibodies in unpurified or partially purified form (i.e., hybridomasupernatant, ascites, polyclonal antisera) or in purified form. A“purified” antibody is one that is separated from at least about 50% ofthe proteins with which it is initially found (i.e., as part of ahybridoma supernatant or ascites preparation). Preferably, a purifiedantibody is separated from at least about 60%, 75%, 90%, or 95% of theproteins with which it is initially found. Suitable derivatives mayinclude fragments (i.e., Fab, Fab₂ or single chain antibodies (Fv forexample)), as are known in the art. The antibodies may be of anysuitable origin or form including, for example, murine (i.e., producedby murine hybridoma cells), or expressed as humanized antibodies,chimeric antibodies, human antibodies, and the like.

Methods of preparing and utilizing various types of antibodies arewell-known to those of skill in the art and would be suitable for use(see, for example, Harlow, et al. Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; Harlow, et al. Using Antibodies: ALaboratory Manual, Portable Protocol No. 1, 1998; Kohler and Milstein,Nature, 256:495 (1975)); Jones et al. Nature, 321:522-525 (1986);Riechmann et al. Nature, 332:323-329 (1988); Presta (Curr. Op. Struct.Biol., 2:593-596 (1992); Verhoeyen et al. (Science, 239:1534-1536(1988); Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al.,J. Mol. Biol., 222:581 (1991); Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,147(1):86-95 (1991); Marks et al., Bio/Technology 10, 779-783 (1992);Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368 812-13(1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995); as well as U.S. Pat. Nos.4,816,567; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and,5,661,016). In certain applications, the antibodies may be containedwithin hybridoma supernatant or ascites and utilized either directly assuch or following concentration using standard techniques. In otherapplications, the antibodies may be further purified using, for example,salt fractionation and ion exchange chromatography, or affinitychromatography using Protein A, Protein G, Protein A/G, and/or Protein Lligands covalently coupled to a solid support such as agarose beads, orcombinations of these techniques. The antibodies may be stored in anysuitable format, including as a frozen preparation (i.e., about −20° C.or −70° C.), in lyophilized form, or under normal refrigerationconditions (i.e., about 4° C.). When stored in liquid form, it ispreferred that a suitable buffer such as Tris-buffered saline (TBS) orphosphate buffered saline (PBS) is utilized. Antibodies and theirderivatives may be incorporated into compositions described herein foruse in vitro or in vivo. Other methods for making and using antibodiesavailable to one of skill in the art may also be suitable for use.

Also provided herein are kits for detecting the presence ofstreptococcus infection in a patient by detecting antibodies or nucleicacid in a biological sample of the patient. In one embodiment, one ormore antigens (e.g., mPLY polypeptides and/or fragment thereof) may formpart of a kit for detecting or diagnosing anti-streptococcal antibodiesin a biological sample. The antigens may be provided in a suitablecontainer such as a vial in which the contents are protected from theexternal environment. Thus, a kit for detecting an anti-streptococcalantibody in a sample may comprise one or more mutant PLY polypeptidesand/or fragments thereof along with one or more detection reagents fordetermining binding of one or more antibodies in a sample to the antigenis provided. The kit preferably includes: (i) one or more isolated andpurified mPLY polypeptides and/or fragments thereof; and, (ii) a systemfor detecting the formation of an antigen-antibody complex, optionallypackaged with instructions for use. The antigen may be free in solutionor may be immobilized on a solid support, such as a magnetic bead, tube,microplate well, or chip. In certain embodiments, a solid matrixcomprising an isolated and purified mPLY polypeptides and/or fragmentsthereof or a fusion protein or protein aggregate adsorbed thereto isprovided. In some embodiments, the kit may further comprise anantibody-binding molecule as a detection reagent. The antibody-bindingmolecule may be a capture or detection reagent and may be free insolution or may be immobilized on a solid support, such as a magneticbead, tube, microplate well, or chip. The antibody-binding molecule orpolypeptide may be labeled with a detectable label, for example afluorescent or chromogenic label or a binding moiety such as biotin.Suitable labels are described in more detail above. The kit may furthercomprise detection reagents such as a substrate, for example achromogenic, fluorescent or chemiluminescent substrate, which reactswith the label, or with molecules, such as enzyme conjugates, which bindto the label, to produce a signal, and/or reagents forimmunoprecipitation (i.e., protein A or protein G reagents). Thedetection reagents may further comprise buffer solutions, washsolutions, and other useful reagents. The kit may also comprise one orboth of an apparatus for handling and/or storing the sample obtainedfrom the individual and an apparatus for obtaining the sample from theindividual (i.e., a needle, lancet, and collection tube or vessel). Thekit may also include instructions for use of the antigen, e.g. in amethod of detecting anti-streptococcal antibodies in a test sample, asdescribed herein. Where the assay is to be combined with another type ofassay such as PCR, the required reagents for such an assay (i.e.,primers, buffers and the like) along with, optionally, instructions forthe use thereof, may also be included.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a”, “an”, and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to a fragment may include mixtures of fragments and referenceto a pharmaceutical carrier or adjuvant may include mixtures of two ormore such carriers or adjuvants.

The terms “about”, “approximately”, and the like, when preceding a listof numerical values or range, refer to each individual value in the listor range independently as if each individual value in the list or rangewas immediately preceded by that term. The terms mean that the values towhich the same refer are exactly, close to, or similar thereto.

As used herein, a subject or a host is meant to be an individual. Thesubject can include domesticated animals, such as cats and dogs,livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratoryanimals (e.g., mice, rabbits, rats, guinea pigs) and birds. In oneaspect, the subject is a mammal such as a primate or a human.

Optional or optionally means that the subsequently described event orcircumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase optionally the composition cancomprise a combination means that the composition may comprise acombination of different molecules or may not include a combination suchthat the description includes both the combination and the absence ofthe combination (i.e., individual members of the combination).

Ranges may be expressed herein as from about one particular value,and/or to about another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent about or approximately, it willbe understood that the particular value forms another aspect. It will befurther understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. Ranges (e.g., 90-100%) are meant to include therange per se as well as each independent value within the range as ifeach value was individually listed.

When the terms prevent, preventing, and prevention are used herein inconnection with a given treatment for a given condition (e.g.,preventing infection by Streptococcus sp.), it is meant to convey thatthe treated patient either does not develop a clinically observablelevel of the condition at all, or develops it more slowly and/or to alesser degree than he/she would have absent the treatment. These termsare not limited solely to a situation in which the patient experiencesno aspect of the condition whatsoever. For example, a treatment will besaid to have prevented the condition if it is given during exposure of apatient to a stimulus that would have been expected to produce a givenmanifestation of the condition, and results in the patient'sexperiencing fewer and/or milder symptoms of the condition thanotherwise expected. A treatment can “prevent” infection by resulting inthe patient's displaying only mild overt symptoms of the infection; itdoes not imply that there must have been no penetration of any cell bythe infecting microorganism.

Similarly, reduce, reducing, and reduction as used herein in connectionwith the risk of infection with a given treatment (e.g., reducing therisk of a pneumococcal infection) typically refers to a subjectdeveloping an infection more slowly or to a lesser degree as compared toa control or basal level of developing an infection in the absence of atreatment (e.g., administration or vaccination using mPLY). A reductionin the risk of infection may result in the patient's displaying onlymild overt symptoms of the infection or delayed symptoms of infection;it does not imply that there must have been no penetration of any cellby the infecting microorganism.

All references cited within this disclosure are hereby incorporated byreference in their entirety. Certain embodiments are further describedin the following examples. These embodiments are provided as examplesonly and are not intended to limit the scope of the claims in any way.

EXAMPLES Example 1 Generation of Expression Plasmids for wtPLY andhis-Tagged wtPLY

The ply gene encoding wtPLY was cloned by PCR using primers Spn001(CATGCCATGGCAAATAAAGCAGTAAATGAC; SEQ ID NO. 45) and Spn002(CAGCCGCTCGAGCTAGTCATTTTCTACCTTATCCTC; SEQ ID NO. 46) from S. pneumoniaestrain R36A (SEQ ID NO. 1, GenBank Accession No. M17717). The PCRproduct was digested with restriction enzymes NcoI and XhoI and clonedinto plasmid pTrcK. Plasmid pTrcK is a kanamycin-resistant derivative ofplasmid pTrcHis2 (Invitrogen). The plasmid thus generated (pBM46) wasused for expression of wtPLY (producing a non-tagged PLY polypeptide)under control of the trc promoter. The DNA sequence of the amplicon wasidentical to the published ply sequence shown in SEQ ID NO. 1), encodingthe deduced amino acid sequence shown in SEQ ID NO. 2. ThewtPLY-encoding nucleic acid was then transferred into pET-28a (EMDBiosciences Cat. No. 69864-3) (NcoI-XhoI), resulting in plasmid pJMS102(providing non-tagged wtPLY expression from the T7 promoter). The samegene was then cloned by PCR using primers 14913.JY(GAAGGAGATATCATATGGCAAATAAAGCAG; SEQ ID NO. 47) and 14914.JY(CCTTTCGGGCTTTGTTAGCAGC; SEQ ID NO. 48) from plasmid pJMS102 back intopET-28a (NdeI-XhoI), resulting in plasmid pJMS112 (providing His-taggedwtPLY expression from the T7 promoter).

Example 2 Generation of Expression Plasmids for PLY(T65C, G293C, C428A)(PlyD1) and His-Tagged PlyD1

Plasmid pJMS112 from Example 1 was used as the template forsite-directed mutagenesis to modify the expressed PLY polypeptide.Site-directed mutagenesis was performed using the QuickChange MultiSite-Directed Mutagenesis kit as per the manufacturer's instructions(Agilent Technologies, Stratagene Products Division, La Jolla, Calif.).The following modifications were introduced into the PLY encodingnucleotide sequence:

the codon for Thr65 was changed from ACC to TGC, encoding cysteine (C)in place of threonine (T) (T65C);

the codon for Gly293 was changed from GGG to TGC, encoding cysteine (C)in place of glycine (G) (G293C); and,

the codon for Cys428 was changed from a TGT to GCT, encoding alanine (A)in place of cysteine (C) (C428A).

The plasmid resulting from the site-directed mutagenesis was designatedpJAY7, and provided for expression of His-tagged PLY(T65C, G293C, C428A)(His-PlyD1). Plasmid pJAY7 was digested with NdeI and XhoI to isolatethe 1420 bp ply gene therefrom. This 1420 bp fragment was gel purifiedand then ligated into NdeI-XhoI cut and dephosphorylated pET-30a (EMDBiosciences Cat. No. 69909-3). The resulting plasmid was named pJMS140,and provided non-tagged PlyD1 expression from the T7 promoter. Thesequence of the mutated ply gene encoding PlyD1 was confirmed using theprimers shown in Table 3 below.

TABLE 3 Primer SEQ ID Name Sequence 5′→3′ NO. T7 TAATACGACTCACTATAGGG 49Promoter 7294.BB GCTAGTTATTGCTCAGCGG 50 13002.MP CTGCTTTTGAAGCTTTGATA 5113003.MP AGGCTTGGGACAGAAATGGG 52 13005.MP TTGAAAGGTCGCAACTACAT 5313006.MP AAACACATCTCCTGGATTTT 54 13007.MP ACTACGAGAAGTGCTCCAGG 55Sequencing of the mutated ply sequence using the primers of Table 3confirmed the mutated sequences were as expected. Expression of PlyD1was confirmed by chemical transformation of E. coli BL21(DE3) withplasmid pJMS140. PlyD1 with a molecular weight of approximately 55 kDawas expressed.

Example 3 Purification of PlyD1 PLY (T65C, G293C, C428A)

To obtain larger quantities of the PlyD1 (SEQ ID NO. 44; T65C, G293C,C428A) was expressed as a recombinant protein (˜55 kDa) in pET30Aplasmid using an E. coli expression system. The E. coli expressedrecombinant PlyD1 protein was grown in a 20-L fermentor. The cells werehomogenized at high pressure to release the soluble PlyD1 anddiafiltered in Tris buffered saline to produce a crude extract of PlyD1.The crude PlyD1 extract was 0.2 um filtered prior to use. The filtered,crude extract was run through the strong anion exchange Giga Cap Q-650 Mcolumn (Tosoh Biosciences). Unbound contaminant proteins were removed inthe flow-through and chased with equilibration buffer (20 mM Tris-HCl,pH 8.5). An intermediate wash step was performed using equilibrationbuffer containing 100 mM NaCl. Bound PlyD1 was eluted with 20 mMTris-HCl, pH 8.5 containing 250 mM NaCl. PlyD1 material recovered fromthis column chromatography step was then conditioned with 5M NaClsolution to bring the conductivity up to −80 mS/cm.

The conditioned PlyD1 material was then subjected to a hydrophobiccolumn chromatography using Phenyl Sepharose FF (GE Health Care). PlyD1was purified using binding and elution mode. Unbound proteins wereremoved by washing the column with 1M NaCl equilibration buffer andbound PlyD1 eluted with the 20 mM Tris-HCl buffer, pH 8.0

The phenyl sepharose-purified PlyD1 material was then diluted 4-foldwith 5 mM sodium phosphate (pH 6.2) to decrease the conductivity and pHof the binding solution. This material was then further purified usingCeramic Hydroxyapatite type I 40 um resin (BioRad). The equilibrationbuffer is 5 mM Sodium Phosphate, pH 6.2. PlyD1 was purified a secondtime using a binding and elution mode on this mixed mode columnchromatography resin. Contaminants were removed using 5 mM sodiumphosphate, pH 7.0, 750 mM NaCl and bound PlyD1 eluted with 10 mM sodiumphosphate, pH 7.0, containing 1 M NaCl.

After the three column steps, purified PlyD1 was diafiltered in 10 mMTris-HCl, pH 7.4, 150 mM NaCl buffer. Tween-80 was added to a finalconcentration of 0.05% to prevent PlyD1 precipitation. The purifiedPlyDI bulk material was 0.2 μM filtered and subsequently formulated atdifferent concentrations. A typical purification process yields 600-850mg/L of purified PlyD1 protein with a purity of >98% based on SDS-PAGEanalysis (FIG. 4).

Example 4 In Vitro Hemolytic Assay

Cytolytic activity of PLY polypeptides is customarily evaluated by an invitro hemolysis assay. Generally, test proteins are serially dilutedacross a plastic microtiter plate in 2-fold serial dilutions with ahighest concentration of 0.5 mg/mL of test protein. BSA is included toprevent adsorptive losses on the plastic microtiter plate. Sheep redblood cells are added to all wells, and the plastic microtiter platesare incubated for 30 min. Lysed cells release hemoglobin. For a positivecontrol, 100% lysis measurement is obtained by the addition of 1%Trition X-100. For a negative control, the sheep red blood cells areincubated with PBS alone. The mictotiter plate is centrifuged toseparate the intact cells from the lysed cells. The supernatantcontaining the lysed cells are transferred to a fresh plate and subjectto an A₅₄₀ hemoglobin release assay. The specific activity is determinedas the inverse of protein concentration (mg/mL) at which 50% hemolysishas occurred relative to the positive control. A representativehemolytic assay system is shown below:

-   -   1. Add 50 μL Hank's buffered saline solution (HBSS)+0.5% (w/v)        bovine serum albumin (BSA) to all the wells of a 96 well        round-bottomed microtiter plates.    -   2. Dilute stock of test protein (e.g., PlyD1) and a negative        irrelevant protein control to a final concentration of 1 mg/mL        with HBSS+0.5% BSA.    -   3. Prepare a 3% sheep red blood cell (RBC) suspension from 10%        stock of washed pooled cells (Rockland Immunochemical, Catalog        number R405-0050) by dilution with HBSS+0.5% BSA.    -   4. Add 50 μL of 1 mg/mL test protein (e.g., PlyD1) (and buffer        and irrelevant protein control) to plate 1 column 2, and        serially dilute (1:2) across two rows (columns 2-12 in 2 rows).        The range of protein concentrations covered will therefore be        from 500 μg/mL to 0.000238 μg/mL. Column 1 is reserved for        buffer blanks (see step 7 below), negative controls (see step 6        below), and 100% lysis control (see step 5 below).    -   5. Setup of 100% lysis controls: Remove 10 μL of HBSS+0.5% BSA        from column 1/rows A-C. Add 10 μL of 10% Triton X-100 in        HBSS+0.5% BSA and 50 μL of 3% sheep RBC solution (from #3 above)        to each of those wells.    -   6. Setup of negative controls: Add 50 μL of either 1 or 3% sheep        RBC solution (from step 3 above) to column 1/rows D-F.    -   7. Setup of buffer blanks: Add 50 μL HBSS+0.5% BSA to column        1/rows G-H.    -   8. Initiation of reaction: Add 50 μL of 3% sheep RBC solution        (from step 3 above) to all of the test wells.    -   9. Incubate for 30 min at 37° C. with rotary shaking    -   10. Add 50 μL of HBSS to all wells and centrifuge for 5 min at        1050 g to spin down non-lysed RBC. (Additional saline        facilitates subsequent step of withdrawing supernatant without        disturbing pellet.)    -   11. Transfer 80 μL of supernatants to a flat-bottomed microtiter        plate, and read absorbances at 540 nm. Plates are blanked with        the buffer blanks from step 7. Percent hemolysis is calculated        by dividing the absorbance in each test well by the average        absorbance of the 100% lysis controls from step 5.    -   12. The data are plotted as percent hemolysis versus protein        concentration (log scale). To determine the specific activity in        hemolytic units per milligram of protein (HU/mg): i) determine        the protein concentration at the 50% hemolysis point, ii)        convert the protein concentration to mg/mL, and iii) take the        inverse of the protein concentration.

This system was used to compare the haemolytic activity of the wild-typeand mutated PLY polypeptides. Table 4 provides a comparison of the invitro hemolytic activity (toxin mediated lysis of sheep red blood cells)of wtPLY and PlyD1 (<0.001%). As shown therein, the mutated PlyD1polypeptide exhibits significantly decreased haemolytic activity ascompared to the wild-type PLY polypeptide.

Example 5 Generation of Anti-PlyD1 Antibodies and Assessment ofAntibody-Mediated Inhibition of wtPLY Activity

PlyD1 was expressed in E. coli and purified as described above. Thepurified proteins were dialyzed against PBS and used for theimmunization of rabbits. Two rabbits were immunized intra-muscularly at20 μg dose with Freund's adjuvant. The animals received two additionalimmunizations with incomplete Freund's adjuvant two and four weeks afterthe initial injection. Serum was collected two and four weeks after thelast injection. The generation of antibodies in the rabbit was firsttested by western blot to ensure that anti-PlyD1 antibodies react withwtPLY. Rabbit sera may also be tested by ELISA and/or IGEN competitionassay using standard procedures to detect the presence of antibodiesspecific for known neutralizing epitopes. Rabbits immunized with PlyD1produced IgG antibodies that bind to full length PLY in ELISA and IGENassays with good serum titres. Thus, production of antibodies reactingwith wtPLY following immunization with PlyD1 was determined.

Generally, the assay to determine the ability of anti-PlyD1 antibodiesto inhibit wtPLY hemolytic activity involves the following steps. First,antisera are produced essentially as described herein then seriallydiluted two-fold across a microtiter plate. A constant amount of nativewtPLY is added to all wells. Sheep red blood cells are added to allwells and the assay proceeds as per the in vitro hemolysis assaydescribed above. After final plate processing, the data are graphed todetermine the 50% inhibition titer. A representativehaemolysis-inhibition assay system is shown below:

-   -   1. Pre-treat antisera to deplete cholesterol (which is        inhibitory to PLY-mediated hemolysis) as follows:        -   a. prepare stock solutions of 20 mg/mL solution of dextran            sulfate (Dextralip 50) and 1 M MgCl₂        -   b. prepare the working solution by mixing equal volumes of            both stock solutions listed in (a) above        -   c. add working solution from (b) above to sera in a 1:10            ratio (i.e. 200 μL working solution to 2 mL sera) and mix            thoroughly for 10 sec        -   d. incubate at room temperature for 15 min, then centrifuge            at 1500×g for 30 min and transfer supernatant to a fresh            tube (cholesterol is found in the precipitate)    -   2. Add 50 μL Hank's buffered saline solution (HBSS)+0.5% (w/v)        bovine serum albumin (BSA) to all the wells of a 96 well        round-bottomed microtiter plates.    -   3. Add 50 μL of each serum to be tested to a separate row in        column 2, and serially dilute (1:2) across the entire row.        Column 1 is reserved for buffer blanks, and positive and        negative controls.    -   4. Prepare a dilution of purified wild-type PLY (non-tagged, lot        DC5826) such that 25 uL contains 10 ng of protein (using        HBSS+0.5% BSA).    -   5. Add 25 uL (containing 10 ng) of diluted wild-type PLY to all        wells except column 1 and allow to incubate with diluted        antisera for 30 min at 37 degrees C. with shaking at 250 rpm.    -   6. Prepare a 3% sheep red blood cell (RBC) suspension from 10%        stock of washed pooled cells (Rockland Immunochemical, Catalog        number R405-0050) by dilution with HBSS+0.5% BSA.    -   7. Setup of 100% lysis controls: Add 15 μL of HBSS+0.5% BSA and        10 μL of 10% Triton X-100 in HBSS+0.5% BSA to column 1/rows A        and B. Add and 25 μL of 1 or 3% sheep RBC solution (from #6        above) to each of those wells.    -   8. Setup of positive controls: Add 25 μL of diluted wild-type        Ply (from #4 above) to column 1/rows C and D. Add and 25 μL of        3% sheep RBC solution (from #6 above) to each of those wells.    -   9. Setup of negative controls: Add 25 μL HBSS+0.5% BSA and 25 μL        of 3% sheep RBC solution (from #6 above) to column 1/rows E and        F.    -   10. Setup of buffer blanks: Add 50 μL HBSS+0.5% BSA to column        1/rows G and H.    -   11. Initiation of reaction: Add 25 μL of 3% sheep RBC solution        (from #6 above) to all of the test wells.    -   12. Incubate for 30 min at 37° C. with shaking at 250 rpm.    -   13. Centrifuge for 5 min at 1050 g to remove non-lysed RBC.    -   14. Transfer 75 μL of supernatants to a flat-bottomed microtiter        plate, and read absorbances at 540 nm. Plates are blanked with        the buffer blanks from step 10. Percent hemolysis is calculated        by dividing the absorbance in each test well by the average        absorbance of the 100% lysis controls from step 7.

This system was used to test the antibodies produced followingimmunization with PlyD1. The data are plotted as percent hemolysisversus antiserum titer (log scale) and the he 50% hemolysis inhibitiontiter is taken as the inverse of the serum dilution at which the percenthemolysis is reduced to 50%. As shown in Table 4 therein, the 50%hemolysis inhibition titer of PlyD1 and the mPLYs modified at G293 (tothreonine, valine or cysteine) is similar to wtPLY. A summary of thecharacteristics observed for PlyD1 polypeptide is provided in Table 4.

TABLE 4 50% hemolysis inhibition titer Hemolytic activity (% Test 1 Test2 vs. wtPLY) Prebleed 4 4 NA Anti-wtPLY 256-512 2048     100% Anti-PdB256-512 ND       1%* Anti-PlyD1 256-512 ND <0.001% (T65C, G293C, C428A)Anti-Ply ND 4096 <0.001% (G293T/V/C) *Observed value for his-tagged PdBwas about 0.5%; observed values for his-tagged wtPLY, and PlyD1 wereabout equivalent to the corresponding untagged polypeptides.FIG. 2 shows that other PLY mutants, including Ply-G293A (G293substituted by alanine), Ply-G293T (G293 substituted by threonine),Ply-G293V (G293 substituted by valine), and Ply-G293C (G293 substitutedby cysteine) also showed lower hemolytic activity (including nodetectable activity) than wtPLY (“Ply” in FIG. 4). Additionalindependent lots of PlyD1 were also tested. Hemolytic activity was notdetected in any of the PlyD1 lots.

Example 6 Immunogenicity of PlyD1 and Adjuvantation

To investigate the immunogenicity of varying doses of PlyD1, eitheralone or in presence of AlOOH adjuvant or stored at differenttemperature to evaluate its stability. Two studies were performed inmice using PlyD1 as an immunogen. In a first study, PlyD1 wasadministered in varying doses, with or without aluminum hydroxide(AlOOH) adjuvant. PlyD1 was found to induce high anti-PLY IgG titers andhemolysis inhibition (HI) titers at all doses tested.

In a second study, PlyD1 was administered with AlOOH adjuvant followingtwo-weeks storage at −70° C., 2-8° C., or 45° C. PlyD1 was administeredin varying doses, with or without AlOOH adjuvant. Mice were immunizedthree times, three weeks apart, and blood samples were taken three weeksafter each immunization. Antibody titers from the second and finalbleeds (three weeks following last immunization) were measured usingPLY-specific ELISA. ELISA titers showed a dose response for anti-PLYtiters following the second and third bleed. Immunization withadjuvanted PlyD1 resulted in significantly higher anti-PLY titerscompared to those induced by unadjuvanted PlyD1. ELISA results from thethird bleed are shown in Table 5.

TABLE 5 PLY-specific Antibody Titers for Groups of Mice Immunized withPlacebo or Increasing Amount of PlyD1, with or without AlOOH ELISA GroupBleed* Mean Titer PlyD1-adjuvanted Pre- <100 (l μg) immunization Post3^(rd) 16890 immunization PlyD1-adjuvanted Pre- <100 (2.5 μg)immunization Post 3^(rd) 20319 immunization PlyD1-adjuvanted Pre- <100(5 μg) immunization Post 3^(rd) 44572 immunization PlyD1-adjuvanted Pre-<100 (10 μg) immunization Post 3^(rd) 41587 immunization PlyD1 Pre- <100(1 μg) immunization Post 3^(rd) 2934 immunization PlyD1 Pre- <100 (2.5μg) immunization Post 3^(rd) 1393 immunization PlyD1 Pre- <100 (5 μg)immunization Post 3^(rd) 6859 immunization PlyD1 Pre- <100 (10 μg)immunization Post 3^(rd) 5572 immunization

In this second study, the ability of antisera from PlyD1-immunized miceto inhibit PLY-mediated hemolysis of sheep red blood cells was testedusing the HI assay following the third bleed was also assessed. Whilethe HI titers appeared to be slightly higher in mice immunized withadjuvanted PlyD1 versus unadjuvanted PlyD1, this difference isconsidered to be within the assay variation and is therefore notsignificant. Furthermore, there did not appear to be a dose response inthe HI titers generated with increasing amounts of PlyD1. Results areshown in Table 6.

TABLE 6 Hemolysis Inhibition Titers in Sera of Mice Immunized with PlyD1with or without AlOOH Prebleed Bleed III Fold Increase Immunization HITiter* HI Titer in HI Titer PlyD1-adjuvanted (1 μg) 2 64 32PlyD1-adjuvanted (2.5 μg) 2 64 32 PlyD1-adjuvanted (5 μg) 2 64 32PlyD1-adjuvanted (10 μg) 2 64 32 PlyD1 (1 μg) 2 16 8 PlyD1 (2.5 μg) 2 3216 PlyD1 (5 μg) 2 32 16 PlyD1 (10 μg) 2 32 16 *HI Titer: highest serumdilution able to completely inhibit hemolysis of a given quantity ofrecombinant wild-type PLY. The lower limit of detection of the assay is4; titers lower than 4 are listed as 2 for statistical purposes.As shown herein, PlyD1 generated high anti-PLY IgG titers when PlyD1,stored at either −70° C. or 2-8° C. was used for immunization, but notPlyD1 stored at 45° C. Moreover, in the presence of adjuvant, PlyD1 wasable to generate higher titers of anti-PLY-specific antibodies incomparison to PlyD1 administered without adjuvant.

These studies show that immunization with PlyD1 elicited neutralizingantibodies at all PlyD1 doses tested. Furthermore, the data indicatethat the immune response to PlyD1 benefits from an adjuvant bygenerating quantitatively more neutralizing antibodies against PLY.

Example 7 TLR4 Assay

wtPLY is known to activate macrophages to generate cytokines (Malley, etal. PNAS USA, 100(4):1966-71 (2003)). This assay determines macrophageactivation by PlyD1 by measuring PlyD1-induced cytokine production invitro. J774 (mouse) and MM6 (human) macrophage-like cells were incubatedovernight with PdB, PlyD1 or PLY treated (+/+) or not treated (−/−) withproteinase K and heat. Heat was used in order to distinguish any falsepositive results due to lipopolysaccharide (LPS) contamination in thetreated group. Cytokine production (IL-6, IL-10, TNF-α, IL-1β) wasmeasured by ELISA after overnight incubation. No induction of cytokines(IL-1β, IL-6 and IL-10) was detected following co-culture of mouse orhuman macrophage cell lines (J774A.1 or MM6, respectively) with PlyD1either untreated or treated with proteinase K/heat. In comparison,untreated wtPLY was able to induce low amounts of IL-1β and IL-6cytokine release in MM6 cells (PLY −/−).

Example 8 Immunogenicity of PlyD1 In Vivo

In testing the immunogenicity of PlyD1 in the models described below,IgG responses elicited by PlyD1 or PdB were tested by ELISA usingrecombinant wtPLY as the coating antigen. A standard assay protocol wasused.

A. Sepsis Model

The ability of PlyD1 to induce anti-PLY antibodies and induce aprotective immune response in mice was assessed. Groups of female BALB/cmice (N=15 per group) were immunized subcutaneously (SC) with purifiedrecombinant PlyD1 or PdB at varying doses, formulated in TBS-containingaluminum adjuvant (260 μg/dose). PdB was administered at 10 μg/dose andPlyD1 at 1 μg, 2.5 μg, 5 μg, and 10 μg/dose. The injection volume was200 μL per dose. TBS placebo-containing aluminum adjuvant was injectedinto negative control groups. Animals were immunized SC at 0, three andsix weeks following initiation of the study. At nine weeks, animals werechallenged by intranasal (IN) injection with 10⁷ colony forming units(cfu) of S. pneumoniae serotype 6B in phosphate-buffered saline (PBS)suspension (50 μL challenge volume per mouse). Following the challenge,mice were monitored for mortality daily. Fourteen days post-challenge,all surviving mice were euthanized. The Fisher Exact Test was used todetermine if there was a significant difference between the immunizedgroup(s) and the placebo control. In addition, sample bleeds were takenfrom all animals following the second boost (day 42) and prior tochallenge, following three immunizations (day 63). Sera were analyzedfor total pneumolysin-specific IgG response by means of an antibodyELISA and for pneumolysin neutralizing capacity in an inhibition ofhemolysis assay.

The differences in time-to-death between mice injected with vaccines andmice injected with placebo were assessed using survival distributionfunctions with product-limit approach. Although antibodies were inducedas shown below, using this sepsis model, mice vaccinated with PlyD1 didnot show delays in time-to-death compared with placebo group, based onWilcoxon test (p=0.5458 for 1 μg, p=0.5003 for 2.5 μg, p=0.1448 for 5μg, and p=0.1723 for 10 μg). Although there was an apparent dose effectwith PlyD1, none of the groups immunized with PlyD1 showed significantsurvival or delay to death compared to the placebo control group(p>0.05) (Table 7).

TABLE 7 Percent Survival of Mice Immunized with Placebo, PdB or PlyD1and Challenged with Serotype 6B Placebo PdB PlyD1 PlyD1 PlyD1 PlyD1 Day(PBS) (10 μg) (1 μg) (2.5 μg) (5 μg) (10 μg) 0 100 100 100 100 100 100 1100 100 100 100 100 100 2 60 100 80 80 93 86 3 60 100 80 80 93 86 4 6093 80 60 93 79 5 60 93 67 53 73 79 6 60 80 60 53 60 71 7 40 67 47 47 6064 8 40 67 47 47 60 64 9 40 67 47 47 60 57 10 40 67 47 47 60 57 11 33 6740 47 60 57 12 33 67 40 47 60 57 13 33 67 40 47 60 57 14 33 67 40 47 6057 p-value¹ survival 0.0716 0.5000 0.3552 0.1362 0.1804 p-value² delay0.0411 0.5458 0.5003 0.1448 0.1723 to death ¹p value was determinedaccording to the fisher exact test and test groups were compared toplacebo control. ²p value was determined according to the Wilcoxon testand test groups were compared to placebo control.

Under this sepsis model, ELISA was performed on days 42 and 63 postimmunization. Results showed high titers of anti-PLY antibodies at alldoses tested. On day 42 and 63, all immunized mice had antibodyresponses against wtPLY, but a significant increase in antibody titerwas not observed after the third vaccination. The anti-PLY IgG titerswere not dose dependent. Pneumolysin-specific antibody titers in the PBSplacebo-, PdB- and PlyD1-immunized groups are summarized in Table 8following the third immunization. Functional antibody titers as measuredby inhibition of hemolysis were higher at day 63 compared to day 42 inall the groups tested (data not shown).

TABLE 8 PLY-Specific Antibody Titers for Groups of Mice Immunized withPlacebo, PdB or PlyD1 ELISA Mean Group Bleed¹ Titer PBS PlaceboPre-immunization <100 Pre-challenge <100 PdB (10 μg) Pre-immunization<100 Pre-challenge 43702 PlyD1 (1 μg) Pre-immunization <100Pre-challenge 87968 PlyD1 (2.5 μg) Pre-immunization <100 Pre-challenge69066 PlyD1 (5 μg) Pre-immunization <100 Pre-challenge 66912 PlyD1 (10μg) Pre-immunization <100 Pre-challenge 76837 ¹Pre-challenge anti-PLYtiters were determined for individual mice and are represented as thegeometrical mean.

As shown herein, using this sepsis model, immunization with recombinantPlyD1 protein generated specific IgG responses but did not showsignificant protection against lethal IN challenge with a S. pneumoniaeserotype 6B strain. However, sera from mice immunized with PlyD1demonstrated PLY-neutralizing capacity.

B. Focal Pneumonia Model

PlyD1 was also tested using a focal pneumonia mouse model. Briefly,groups of 10 CBA/N mice were immunized SC with purified recombinantPlyD1 proteins at variable doses formulated in TBS-containing aluminumadjuvant (300 μg/dose). The injection volume was 200 μL per dose.Phosphate-buffered saline placebo-containing aluminum adjuvant wasinjected into negative control groups. Animals were immunized SC threetimes at 0, three and six weeks following initiation of the study. Threeweeks after the last immunization, animals were challenged intranasally(IN) with 3-7×10⁶ cfu of S. pneumoniae strain EF3030 (serotype 19F; 40μL challenge volume per mouse). Mice were sacrificed 5 dayspost-challenge and lung tissue harvested and plated for cfu recovery.The Mann-Whitney Test was used to determine if there was a significantdifference between immunized group(s) and the placebo control group. Instudies using this model, sera analysis for either IgG titer orneutralizing capacity of the sera was not performed. All groups thatwere immunized with PlyD1 did not have significantly lower bacteriallung burden when compared to the PBS-immunized group and thus, wereconsidered as not protected (data not shown).

C. Intranasal Challenge Model

PlyD1 was also evaluated in-house using an intranasal challenge mousemodel. In this model, groups of female CBA/j mice (N=15 per group) wereimmunized IM with purified recombinant PlyD1 proteins at doses rangingfrom 0.25 to 25 μg/dose. PlyD1 was formulated in TBS-containing aluminumadjuvant (65 μg/dose). The injection volume was 50 μL per dose. PBSplacebo-containing aluminum adjuvant was injected into negative controlgroups. Animals were immunized IM at 0, 3, and 6 weeks followinginitiation of the study. At nine weeks, animals were challenged IN witha lethal dose (5×10⁵ cfu) of S. pneumoniae strain 14453 (serotype 6B) inPBS suspension (40 μL challenge volume per mouse). Following thechallenge, mice were monitored daily for mortality. All surviving micewere euthanized 11 days post-challenge. The Fisher Exact Test was usedto determine if there was a significant difference between the immunizedgroup(s) and the placebo control. In addition, sample bleeds were takenfrom all animals 4 days prior to the first injection (pre-immunizationat 0 weeks) and following each immunization. Sera were analyzed fortotal PlyD1-specific IgG responses by means of an antibody ELISA and forpneumolysin neutralizing capacity in an inhibition of hemolysis assay.

PlyD1 was administered at 0.25 μg, 0.5 μg, 1 μg, 5 μg, 10 μg and 25μg/dose or an adjuvant alone (placebo). Mice were immunized three times,three weeks apart and blood samples were taken three weeks followingeach immunization. Antibody titers from the second bleed and final bleed(three weeks following last immunization) were measured usingPLY-specific ELISA. The ability of the first, second and final bleedsera to inhibit PLY-mediated hemolysis was assessed. Mice werechallenged with S. pneumoniae strain 14453 (serotype 6B) at 5×10⁵cfu/dose three weeks following the last immunization and survival wasmonitored for ten days. Survival of mice immunized with PlyD1 wassignificantly better than placebo group at doses of 5, 2.5 and 0.25μg/dose, indicating that protection is not dose-dependent. The bestprotection was observed at 2.5 μg/dose, in which 60% of the micesurvived compared to none in the placebo group. The results aresummarized in Table 9.

TABLE 9 Percent Survival of Mice Immunized with Placebo or PlyD1 atVarious Doses and Challenged with S. pneumoniae Strain 14453 PlaceboPlyD1 PlyD1 PlyD1 PlyD1 PlyD1 PlyD1 PlyD1 Day (AlOOH) (25 μg) (10 μg) (5μg) (2.5 μg) (1 μg) (0.5 μg) (0.25 μg) 0 100 100 100 100 100 100 100 1001 100 100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 100 391.7 100 91.7 100 100 90.1 100 100 4 41.7 83.3 58.3 91.7 66.7 45.5 75 755 8.3 50 41.7 50 58.3 36.4 33.3 58.3 6 8.3 50 33.3 50 58.3 36.4 25 58.37 0 16.7 16.7 41.7 58.3 18.2 16.7 58.3 8 0 16.7 16.7 41.7 58.3 18.2 16.741.7 9 0 16.7 16.7 41.7 58.3 18.2 16.7 41.7 10 0 16.7 16.7 41.7 58.318.2 16.7 41.7 p-value¹ survival 0.239 0.239 0.0466 0.0023 0.26 0.2390.0466 ¹p value was determined according to the fisher exact test andtest groups were compared to placebo control.

The ability of antisera from mice immunized using this intranasalchallenge model was also assessed for inhibition of PLY-mediatedhemolysis of sheep red blood cells. Sera from all bleeds were pooledaccording to their treatment groups and tested using the HI assayfollowing the all bleeds. While the HI titers appeared to be slightlyhigher after the third immunization (bleed 3) in mice immunized with 2.5and 5 μg/dose, this difference is considered to be within the assayvariation and therefore is not significant. Furthermore, there did notappear to be a dose response in the HI titers generated with increasingamounts of PlyD1. The results are shown in Table 10.

TABLE 10 Hemolysis Inhibition Titers in Sera of Mice Immunized withPlyD1 at Different Doses Fold Increase Between HI Titer* Prebleed toImmunogen Prebleed Bleed 1 Bleed 2 Bleed 3 Bleed 3 HI Titer PlyD1 (25ug) 4 8 32 64 16 PlyD1 (10 ug) 4 8 32 64 16 PlyD1 (5 ug) 2 8 64 128 64PlyD1 (2.5 ug) 2 8 32 128 64 PlyD1 (1 ug) 2 4 32 64 32 PlyD1 (0.5 ug) 28 64 64 32 PlyD1 (0.25 ug) 2 4 64 64 32 Placebo 2 2 2 2 1 (AlOOH) *HITiter: highest serum dilution able to completely inhibit hemolysis of agiven quantity of recombinant wtPLY. The lower limit of detection of theassay is 4; titers lower than 4 are listed as 2 for statisticalpurposes.

Antibody titers from the second bleed and third bleed were measuredusing quantitative anti-PLY ELISA. All PlyD1 immunized mice were able tomount an antibody response to PLY following bleed 2 and bleed 3. Therewere no significant differences (<2 fold differences) observed betweengroups immunized with increasing doses of PlyD1, suggesting that alldoses tested were still generating saturating amounts of anti-PLYantibodies. ELISA results from the third bleed are shown in Table 11.

TABLE 11 PLY-Specific Antibody Titers for Groups of Mice Immunized withPlacebo or Increasing Amount of PlyD1 ELISA Mean Group Bleed¹ TiterPlyD1-adjuvanted Pre-immunization <3 (25 μg) Post 3^(rd) immunization35724 PlyD1-adjuvanted Pre-immunization <3 (10 μg) Post 3^(rd)immunization 28900 PlyD1-adjuvanted Pre-immunization <3 (5 μg) Post3^(rd) immunization 58699 PlyD1-adjuvanted Pre-immunization <3 (2.5 μg)Post 3^(rd) immunization 38093 PlyD1-adjuvanted Pre-immunization <3 (1μg) Post 3^(rd) immunization 17765 PlyD1-adjuvanted Pre-immunization <3(0.5 μg) Post 3^(rd) immunization 25047 PlyD1-adjuvantedPre-immunization <3 (0.25 μg) Post 3^(rd) immunization 20883 PlaceboPre-immunization <3 (AlOOH) Post 3^(rd) immunization 26 ¹Performed byanti-PLY quantitative ELISA

Balb/c mice were also immunized with PlyD1 with or without adjuvant,wtPLY at a dose of 5 or 10 μg, and then challenged with a 5 μg dose ofwtPLY. The lungs were then harvested for H&E stain to observe tissuedamage caused by wtPLY. Compared to controls (mice immunized withTris-saline, 15% glycerol), wtPLY typically causes perivascular edema,thickened, disrupted alveolar walls, diminished alveolar space, andfluid and blood infiltration of the alveolar spaces. As shown in FIG. 3,mice immunized with PlyD1, PdB, or wtPLY demonstrated significantprotection from lung damage after intranasal challenge with wtPLY. Theability of antisera from these immunized mice to inhibit PLY-mediatedhemolysis of sheep red blood cells was tested using the HI assayfollowing the first, second and third bleed. Neutralizing antibodytiters were increased following bleed 3 compared to bleed 2. Takentogether, PlyD1 immunization can generate antibodies that are directlyinvolved in the neutralization of PLY toxicity in vivo.

CBA/J mice were immunized (I.M) with adjuvant alone (AlOOH) or withmonovalent Ply-D1, 3 times with 3 weeks interval between eachimmunization. 3 weeks post final immunization, mice were challenged withGT-14453.BM2 (serotype 6B) at 5×10⁵ CFU/mouse and observed forsurvival/health for 2 weeks. PlyD1 was formulated as 0.25 μg, 0.5 μg,1.0 μg, 2.5 μg, 5.0 μg, 10 μg and 25 μg/ml. Data was analyzed byplotting survival/health status of mice and statistically compared usingFisher-one sided test for statistical analysis. As shown in FIG. 5,immunization with recombinant PlyD1 led to protection against lethalchallenge in mice.

As shown herein, using the intranasal bacterial challenge model,significant protection was observed in mice immunized with PlyD1compared to placebo control. PlyD1 mice had significantly lower lungdamage when compared to placebo-immunized and PLY challenged mice.Collectively these data indicate that PlyD1 immunization of mice cangenerate antibodies that are directly involved in the neutralization ofPLY toxicity in vivo as well as protect from a lethal IN challenge usinglive bacteria.

Example 9 Immunogenic Composition and/or Vaccine

The production process for a PlyD1 immunogenic composition and/orvaccine involves: 1) growing recombinant E. coli cells that express thePlyD1 protein in a fermenter, using pH-Stat fed batch fermentation; 2)recovering the PlyD1 protein by homogenizing cells followed by 0.2 μmclarification filtration, and concentration by ultrafiltration; 3)purifying the PlyD1 protein using ion-exchange chromatography andhydrophobic interaction chromatography; and, 4) formulating the purifiedPlyD1 protein with aluminum adjuvant, however other adjuvants known inthe art an also be used. These procedures are described herein orwell-known to those of skill in the art of vaccine formulation.

PlyD1 may be formulated with an aluminum hydroxide adjuvant in sterileTris-buffered saline (TBS) without preservative, and prepared as asterile, white opaque liquid suspension in single-dose vials (containing0.28 mg of elemental aluminum/dose). Other adjuvants known in the artcan also be used to formulate a vaccine. Each 0.5 mL dose of PlyD1immunogenic composition or vaccine typically contains the followingcomponents: recombinant PlyD1 (10 μg (low dose), 25 μg (middle dose), 50μg (high dose)), and Tris-buffered saline (TBS; 10 mM Tris-HCl pH 7.4,150 mM sodium chloride), aluminum hydroxide adjuvant (0.28 mg elementalaluminum/dose), and sodium phosphate (2 mM) to optimize binding andstability of adsorbed antigens. The glass vials are filled with0.72±0.05 mL to give a withdrawable volume of 0.50 mL. An injection(intramuscular, IM) of 0.5 mL from a low dose, middle dose and high dosewill give a dosage of either 10 μg, 25 μg or 50 μg of PlyD1,respectively. Each 0.5 mL dose is adjuvanted with 0.28 mg±0.10 mgelemental aluminum. The placebo to be used is TBS. The immunogeniccomposition or vaccine is typically supplied in 3 mL glass vials thathave a 13 mm gray butyl serum stopper that is latex-free and a 13 mmone-piece aluminum seal. The composition is typically a white cloudysuspension and the placebo is a clear solution and must be stored atabout 2° C. to 8° C. (e.g., not frozen). The aluminum adjuvant in theproduct typically settles over time and is re-suspended before use. Inpreparation for use, the vaccine vial should be inverted about 5 to 10times until the contents are uniform in appearance. A syringe should befilled immediately after suspension/mixing and the vaccine injectedpromptly. Routes of administrations may be as described herein, forexample, preferably via subcutaneous (SC), intradermal (ID),intramuscular (IM), or oral routes.

While certain embodiments have been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the following claims.

SEQUENCE LISTINGSEQ ID NO.: 1 (pBM46 nucleotide sequence wtPLY insert S. pneumoniaeR36A)ATGGCAAATAAAGCAGTAAATGACTTTATACTAGCTATGAATTACGATAAAAAGAAACTCTTGACCCATCAGGGAGAAAGTATTGAAAATCGTTTCATCAAAGAGGGTAATCAGCTACCCGATGAGTTTGTTGTTATCGAAAGAAAGAAGCGGAGCTTGTCGACAAATACAAGTGATATTTCTGTAACAGCTACCAACGACAGTCGCCTCTATCCTGGAGCACTTCTCGTAGTGGATGAGACCTTGTTAGAGAATAATCCCACTCTTCTTGCGGTTGATCGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACCCCAGCAATTCAAGTGTTCGCGGAGCGGTAAACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAGCTAGAATGCAGTATGAAAAAATAACGGCTCACAGCATGGAACAACTCAAGGTCAAGTTTGGTTCTGACTTTGAAAAGACAGGGAATTCTCTTGATATTGATTTTAACTCTGTCCATTCAGGTGAAAAGCAGATTCAGATTGTTAATTTTAAGCAGATTTATTATACAGTCAGCGTAGACGCTGTTAAAAATCCAGGAGATGTGTTTCAAGATACTGTAACGGTAGAGGATTTAAAACAGAGAGGAATTTCTGCAGAGCGTCCTTTGGTCTATATTTCGAGTGTTGCTTATGGGCGCCAAGTCTATCTCAAGTTGGAAACCACGAGTAAGAGTGATGAAGTAGAGGCTGCTTTTGAAGCTTTGATAAAAGGAGTCAAGGTAGCTCCTCAGACAGAGTGGAAGCAGATTTTGGACAATACAGAAGTGAAGGCGGTTATTTTAGGGGGCGACCCAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATGGTAGAGGACTTGATTCAAGAAGGCAGTCGCTTTACAGCAGATCATCCAGGCTTGCCGATTTCCTATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAACAGTACAGACTATGTTGAGACTAAGGTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCATAGTGGTGCCTATGTTGCCCAATATTATATTACTTGGGATGAATTATCCTATGATCATCAAGGTAAGGAAGTCTTGACTCCTAAGGCTTGGGACAGAAATGGGCAGGATTTGACGGCTCACTTTACCACTAGTATTCCTTTAAAAGGGAATGTTCGTAATCTCTCTGTCAAAATTAGAGAGTGTACCGGGCTTGCCTGGGAATGGTGGCGTACGGTTTATGAAAAAACCGATTTGCCACTAGTGCGTAAGCGGACGATTTCTATTTGGGGAACAACTCTCTATCCTCAGGTAGAGGATAAGGTAGAAAATGACTAGSEQ ID NO.: 2 PLY (pBM46)MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDENSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISINGTTLYPQVEDKVEND SEQ ID NO.: 3 (PdB)MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDENSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISINGTTLYPQVEDKVEND SEQ ID NO.: 4 (ΔAlal46)MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPRMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDENSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISINGTTLYPQVEDKVEND SEQ ID NO.: 5 (ΔAla146R147)MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPRRMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 6GenBank EF413923MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALMKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 7GenBank EF413924MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVDAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 8GenBank EF413925MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 9GenBank EF413926MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQHEKITAHSMEQLKVKFGSDFEKIGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLRQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 10GenBank EF413927MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQHEKITAHSMEQLKVKFGSDFEKIGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLRQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 11GenBank EF413928MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQHEKITAHSMEQLKVKFGSDFEKIGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLRQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 12GenBank EF413929MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGEDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 13GenBank EF413930MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 14GenBank EF413931MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVDPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 15GenBank EF413932MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQHEKITAHSMEQLKVKFGSDFEKIGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLRQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 16GenBank EF413933MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHKDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 17GenBank EF413934MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKIMAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 18GenBank EF413935MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKIMAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 19GenBank EF413936MANKAVNDFILAMDYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLRQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 20GenBank EF413937MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKIGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLRQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 21GenBank EF413938MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQHEKITAHSMEQLKVKFGSDFEKIGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLRQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 22GenBank EF413939MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGFDFEKIGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLRQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 23GenBank EF413940MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 24GenBank EF413941MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 25GenBank EF413942MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHHDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 26GenBank EF413943MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 27GenBank EF413944MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 28GenBank EF413945MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 29GenBank EF413946MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 30GenBank EF413947MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 31GenBank EF413949MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 32GenBank EF413950MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 33GenBank EF413951MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 34GenBank EF413952MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 35GenBank EF413953MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 36GenBank EF413954MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 37GenBank EF413955MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDENSVHSGEKQIQIVNEKQTYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYTTWDELSYDHQGKEVLTPKAWDRNGQDLTAHETTSIPLKGNVRNLSVKIRECTGLAWENWRTVYEKTDLPLVRKRTISINGTTLYPQVEDKVEND SEQ ID NO.: 38GenBank EF413956 (ply-10)MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKIMAHSMEQLKVKFGSDFEKTGNSLDIDENSVHSGEKQIQIVNEKQTYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYTTWDELSYDHQGKEVLTPKAWDRNGQDLTAHETTSIPLKGNVRNLSVKIRECTGLAWENWRTVYEKTDLPLVRKRTISINGTTLYPQVEDKVEND SEQ ID NO.: 39GenBank EF413957 (ply-3)MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKIGNSLDIDENSVHSGEKQIQIVNEKQTYYTVSVDAVKNPGDVFQDTVTVEDLRQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYTTWDELSYDHQGKEVLTPKAWDRNGQDLTAHETTSIPLKGNVRNLSVKIRECTGLAWENWRTVYEKTDLPLVRKRTISINGTTLYPQVEDKVEND SEQ ID NO.: 40GenBank EF413958 (ply-5)MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQHEKITAHSMEQLKVKFGSDFEKIGNSLDIDENSVHSGEKQIQIVNEKQTYYTVSVDAVKNPGDVFQDTVTVEDLRQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYTTWDELSYDHQGKEVLTPKAWDRNGQDLTAHETTSIPLKGNVRNLSVKIRECTGLAWENWRTVYEKTDLPLVRKRTISINGTTLYPQVEDKVEND SEQ ID NO.: 41GenBank EF413959 (ply-2)MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDENSVHSGEKQIQIVNEKQTYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYTTWNELSYDHQGKEVLTPKAWDRNGQDLTAHETTSIPLKGNVRNLSVKIRECTGLAWENWRTVYEKTDLPLVRKRTISINGTTLYPQVEDKVEND SEQ ID NO.: 42GenBank EF413960 (ply-5)MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQHEKITAHSMEQLKVKFGSDFEKIGNSLDIDENSVHSGEKQIQIVNEKQTYYTVSVDAVKNPGDVFQDTVTVEDLRQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFESLIKGVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND SEQ ID NO.: 43 (G293C)MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILCGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVENDSEQ ID NO.: 44 (T65C, G293C C428A)MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTACNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILCGDPSSGARVVTGKVDMVEDLIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLTAHFTTSIPLKGNVRNLSVKIREATGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVENDSEQ ID NO.: 45 Spn1, Forward, NcoI CATGCCATGGCAAATAAAGCAGTAAATGACSEQ ID NO.: 46 Spn2, Reverse, XhoI CAGCCGCTCGAGCTAGTCATTTTCTACCTTATCCTCSEQ ID NO.: 47 14913.JY, Forward, Ndel GAAGGAGATATCATATGGCAAATAAAGCAGSEQ ID NO.: 48 14914.JY, Reverse CCTTTCGGGCTTTGTTAGCAGC SEQ ID NO.: 49T7 Promoter TAATACGACTCACTATAGGG SEQ ID NO.: 50 7294.BBGCTAGTTATTGCTCAGCGG SEQ ID NO.: 51 13002.MP CTGCTTTTGAAGCTTTGATASEQ ID NO.: 52 13003.MP AGGCTTGGGACAGAAATGGG SEQ ID NO.: 53 13005.MPTTGAAAGGTCGCAACTACAT SEQ ID NO.: 54 13006.MP AAACACATCTCCTGGATTTTSEQ ID NO.: 55 13007.MP ACTACGAGAAGTGCTCCAGG

1. An isolated polypeptide consisting of a wild-type pneumolysinpolypeptide comprising at least one amino acid substitution at an aminoacid selected from the group consisting of threonine 65, glycine 293,and cysteine
 428. 2. An isolated polypeptide consisting of a wild-typepneumolysin polypeptide comprising at least one amino acid substitutionat threonine 65 and at least one of glycine 293 or cysteine
 428. 3. Thepolypeptide of claim 1 wherein the wild-type pneumolysin has at least90% identity to SEQ ID NO.:3.
 4. The polypeptide of claim 1 wherein thewild-type pneumolysin has at least 95% identity to SEQ ID NO.:3.
 5. Thepolypeptide of claim 1 wherein the wild-type pneumolysin has at least99% identity to SEQ ID NO.:3.
 6. The polypeptide of claim 1 wherein thesubstitution of threonine 65 is by cysteine, the substitution is ofglycine 293 is by an amino acid selected from the group consisting ofcysteine, valine, and threonine, and the substitution of cysteine 428 isby alanine.
 7. An immunogenic composition comprising the polypeptide ofclaim 1 and a pharmaceutically acceptable carrier.
 8. The immunogeniccomposition of claim 7, further comprising at least one additional S.pneumoniae antigen.
 9. The immunogenic composition of claim 8 whereinthe S. pneumoniae antigen is at least one of PcPA or PhtD.
 10. Theimmunogenic composition of claim 9 comprising PcPA and PhtD.
 11. Anisolated nucleic acid sequence encoding the pneumolysin polypeptide ofclaim
 1. 12. An expression vector comprising a nucleic acid sequenceencoding a modified pneumolysin polypeptide of claim
 1. 13. A host cellcomprising with an expression vector encoding a polypeptide of claim 1.14. A host cell transformed, transfected or infected with an expressionvector of claim
 12. 15. A method of producing a polypeptide of claim 1comprising transfecting a host cell with an expression vector encodingthe polypeptide, culturing the host cell such that the polypeptide isexpressed, and isolating the polypeptide.
 16. A method of eliciting animmune response in a mammal, the method comprising administering to themammal a composition comprising the isolated polypeptide of claim 1along with a pharmaceutically acceptable carrier.
 17. A method ofgenerating antibodies that bind to a wild-type pneumolysin polypeptide,the method comprising introducing into a mammal a composition comprisingthe isolated polypeptide of claim 1 along with a pharmaceuticallyacceptable carrier, the composition optionally further comprising anadjuvant.
 18. An isolated antibody reactive with the polypeptide ofclaim
 1. 19. Use of a substantially purified polypeptide of claim 1 inthe manufacture of a medicament for treatment of a bacterial infection.20. A vaccine composition comprising the polypeptide of claim 1.